Gamma-1 and gamma-3 anti-human cd23 monoclonal antibodies and use thereof as therapeutics

ABSTRACT

Monoclonal antibodies which specifically bind human CD23, the low affinity receptor for IgE (FceRII/CD23), and contain either a human gamma-1 or human gamma-3 constant domain, are disclosed. The antibodies are useful for modulating or inhibiting induced IgE expression. Accordingly, they have practical utility in the treatment or prophylaxis of disease conditions wherein inhibition of induced IgE production is therapeutically desirable, including allergic conditions, autoimmune diseases and inflammatory diseases.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. Ser. No. 08/803,085,filed Feb. 20, 1997.

FIELD OF THE INVENTION

The present invention relates to monoclonal antibodies whichspecifically bind human CD23, the low affinity receptor for IgE(FceRII/CD23), and contain either a human gamma-1 or human gamma-3constant domain, and their usage as therapeutic agents.

BACKGROUND OF THE INVENTION

IgE is a member of the immunoglobulin family that mediates allergicresponses such as asthma, food allergies, type 1 hypersensitivity andthe familiar sinus inflammation allergic rhinitis and conjunctivitis,and as a result, causes widespread suffering throughout the generalpopulation. IgE is secreted by, and expressed on the surface of,B-cells. IgE synthesized by B-cells can be anchored in the B-cellmembrane by a short transmembrane domain linked to the mature IgEsequence. Membrane and secreted versions of IgE are formed in the samecell by differential splicing of the IgE RNA transcript.

IgE also can be bound to B-cells (and T cells, monocytes, Langerhanscells, follicular dendritic cells, natural killer cells, eosinophils andplatelets) through its Fc region to a low affinity IgE receptor (FceRII,hereafter “FCEL”, and to mast cells and basophils through its Fc regionto a high affinity IgE receptor FceRI, (hereinafter “FCEH”). The lowaffinity IgE receptor is generally referred to in the literature asCD23.

Upon exposure of a mammal to an allergen, antigen presenting cellsprocess the antigen for presentation to helper T cells. These helper Tcells secrete cytokines such as IL-4 which assist B-cells to undergoclonal amplification and secrete more allergen-specific IgE. This newlysynthesized IgE in turn is released into the circulation where it bindsto mast cells and basophils through the high affinity receptor on theircell surface. Such mast cells and basophils are thereby sensitized tothe specific allergen. The next exposure to the same allergen causesbinding to specific IgE on the surface of mast cells, and basophils,thereby cross-linking the FceRI on these cells and thus activating theirrelease of histamine and other factors which are responsible forclinical hypersensitivity and anaphylaxis.

The art has reported antibodies capable of binding to FCEL (CD23)-boundIgE but not IgE bound to FCEH (see, for example, WO 89/00138 and U.S.Pat. No. 4,940,782). These antibodies are disclosed to be clinicallyadvantageous because they bind to IgE which is bound to the low affinityreceptor (FCEL) or to circulating IgE's, but do not bind to IgE bound tothe high affinity receptor (FCEH). Therefore, these antibodies will notactivate mast cells or basophils.

Moreover, anti-CD23 antibodies have been reported to have potential astherapeutics, e.g., for the treatment of allergic disorders,inflammatory diseases, and autoimmune diseases. For example, Bonnefoy etal., WO 9612741, report that ligands which bind CD23, e.g., monoclonalantibodies, are useful in the treatment or prophylaxis of inflammatory,autoimmune and allergic diseases.

The usage of monoclonal antibodies to CD23, as both IgE agonists andantagonists has been reported. IgE antagonists have been reported tohave potential utility in treatment of conditions or diseases whereinIgE suppression is therapeutically desirable, e.g., allergic conditionssuch as allergic rhinitis and conjuntivitis, atopic dermatitis andasthma. For example, Bonnefoy et al., WO 8707302 (1987), reportmonoclonal antibodies to human CD23, which are assertedly useful forassaying the presence of IgE receptors on cell types and as therapeuticsin diseases wherein modulation of IgE is therapeutically desirable.

In part because of their potential as therapeutics and diagnostics, manygroups have reported the generation of monoclonal antibodies to CD23.See, e.g., Rector et al., Immunol., 55:481-488 (1985); Suemura et al.,J. Immunol., 137:1214-1220 (1986); Noro et al., J. Immunol.,137:1258-1263 (1986); Bonnefoy et al., J. Immunol., 138:2170-2178(1987); Flores-Romo et al., Science, 261:1038-1046 (1993); Sherr et al.;J. Immunol., 142:481-489 (1989); and Pene et al., Proc. Natl. Acad.Sci., USA, 85:6880-6884 (1988). Moreover, as discussed supra, the usageof such antibodies specifically to inhibit IgE production in systemswhere IgE synthesis is cytokine (IL-4) induced has also been reported.(Flores-Romo et al (Id.); Sherr et al. (Id.); Bonnefoy et al. (WO8707302); Bonnefoy et al. (WO 8707302); Bonnefoy et al. (WO 9612741));Bonnefoy et al., Eur. J. Immunol. 20:139-144 (1990); Sarfati et al., J.Immunol 141:2195-2199 (1988) and Wakai et al., Hybridoma 12:25-43(1993). Also, Flores-Romo et al. (Id.) disclose that Fabs prepared fromanti-CD23 antibodies inhibit antigen-specific induced IgE responses invivo in the rat. However, notwithstanding what has been reported, themechanism by which anti-CD23 antibodies modulate IgE expression and inparticular, the manner by which they block IL-4 induced IgE productionremains unclear.

It has been suggested that anti-CD23 antibodies inhibit IgE productionby signaling through CD23 present on the surface of IgE secreting Bcells. It has been proposed that the function of CD23, which isupregulated on IgE secreting B cells, is feedback inhibition of IgEproduction (Yu, et al. Nature 369, 753-756 (1994)). This has beentheorized because mice in which the CD23 gene has been removed haveincreased and sustained IgE production compared to controls (Yu, etal.). In addition, it has been reported that binding to CD23 by IgEcomplexes or by a monoclonal antibody to anti-CD23 suppresses ongoingIgE synthesis by a lymphoblastoid cell line that constitutively secretesIgE (Sherr et al. (Id.)). It appears that this is due to down regulationof the messenger RNA for the secreted IgE heavy chain in this cell(Saxon et al., J. Immunol., 147:4000-4006 (1991)). However, the exactmechanism by which IgE expression is inhibited has yet to be explainedin systems in which IgE secretion is IL-4 induced.

It has also been reported that crosslinking of Fc gamma RII with surfaceIg (B cell receptor) on B cells leads to down regulation of Igexpression. (D'Ambrosia et al., Science, 268:293-297 (1995). A similarmechanism can be proposed for B cells secreting IgE which also have cellsurface CD23 and Fc gamma RII. An anti-human CD23 antibody bound to acell by antigen (CD23) and also bound to Fc gamma RII through Fcinteractions could transmit a signal to suppress IgE secretion throughFc gamma RII.

Mechanisms involved in IgE inhibition by anti-CD23 antibodies have beenproposed that include blocking interactions other than the interactionbetween membrane CD23 and IgE. Related to this, CD23, which is a memberof the C-type lectin family, has been shown to interact with severalother ligands such as CD21, CD11b and CD11c present on a variety of celltypes including T cells and monocytes. In this context CD23 can beenvisioned as a cellular adhesion molecule.

Therefore, it has been proposed that the CD21-CD23 interaction may beinvolved in antigen presentation and subsequent IgE production. Modelssuggest CD21 on B cells sending an activation signal for IgE productionafter binding to CD23 on activated T cells present primarily in atopicindividuals. (Leconant et al., Immunol., 88:35-39 (1996); and Bonnefoyet al., Int. Amer. Allergy Immunol., 107:40-42 (1995).) Blocking thisinteraction with an anti-CD23 could block induced IgE production. (Aubryet al., Nature, 358:505-507 (1992) and Immunol., 5:944-949 (1993);Grosjean et al. (1992); Bonnefoy et al., Curr. Opin. Eur. J. Immunol.,24:2982-2988 (1994); Henchoz-Lecoanet et al., Immunol., 88:35-39 (1996)Nambu et al., Immunol. Lett., 44:163-167 (1995); Bonnefoy et al., Int.Amer. Allergy Immunol., 107:40-42 (1995).) It is also possible thatantigen presentation is upregulated by CD23 on antigen presenting Bcells binding to CD21 on T cells.

Yet another mechanism which would potentially explain the effects ofCD23 on IgE production involves soluble forms of CD23. It has beenreported that CD23 is cleaved from the cell surface releasing severaldifferent forms of soluble CD23 or IgE binding factors. (Sarfati et al.,Immunol., 53:197-205 (1984).) Soluble CD23 is a cytokine, with one ofits reported activities being the augmentation of IL-4 induced IgEproduction from B cells. (Pene et al., J. Cell Biochem., 39:253-269(1989); Pene et al., Eur. J. Immunol., 18:929-935 (1988); Sarfati etal., J. Immunol., 141:2195-2197 (1988); Sarfati et al. (1984) (Id.);(Saxon et al., J. Clin. Immunol. Allergy, 86 (3 pt 1) 333-344 (1990).Also, certain forms of soluble CD23 have been reported to inhibit IgEproduction (Sarfati et al., Immunol., 76:662-667 (1992)). Accordingly,anti-CD23 antibodies potentially may block IgE production by 1)inhibiting the IgE augmenting effects of soluble CD23 and/or 2) blockingthe proteolytic release of soluble CD23 from the cell surface.

Thus, based on the foregoing, it is clear that there is significantcomplexity and uncertainty in the art with respect to the functions ofmore specifically CD23 and effects on IgE production, and further withrespect to the means by which ligands specific thereto affect IgEproduction.

OBJECTS OF THE INVENTION

Thus, it is an object of the invention to produce novel ligands(antibodies) specific to CD23 and to use such antibodies to elucidatethe mechanism by which anti-CD23 antibodies modulate IgE expression.

It is another object of the invention to produce novel ligands(antibodies) which bind CD23, in particular human CD23, having improvedability to inhibit induced IgE expression.

It is a more specific object of the invention to produce anti-human CD23antibodies containing human gamma-1 or human gamma-3 constant domains.

It is another object of the invention to produce multivalent anti-humanCD23 antibodies which may be more effective by virtue of their enhancedpotential for cross linking CD23 and Fc receptors.

It is another object of the invention to provide pharmaceuticalcompositions containing anti-human CD23 monoclonal antibodies comprisinghuman gamma-1 or gamma-3 constant domains which are capable ofinhibiting induced IgE production.

It is another object of the invention to use an anti-human CD23monoclonal antibody comprising a human gamma-1 or human gamma-3 constantdomain for treatment or prophylaxsis of disease conditions whereininhibition of induced IgE production is therapeutically desirable.

More specifically, it is an object of the invention to treat or preventallergic conditions, autoimmune diseases and inflammatory diseases usingan anti-human CD23 monoclonal antibody comprising either a human gamma-1or human gamma-3 constant domain.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 compares the in vitro IgE inhibitory activity of a murineanti-human CD23 monoclonal antibody (MHM6), to five primate anti-humanCD23 monoclonal antibodies (5E8, 6G5, 2C8, B3B11, and 3G12);

FIG. 2 shows that primate monoclonal antibodies 5E8 and 6G5 bind anepitope on human CD23 that is distinct from commercially availablemurine anti-human CD23 monoclonal antibody MHM6 (middle panel, FIG. 2)and compete with each other (lower panel, FIG. 2). Primate anti-humanCD23 monoclonal antibodies 2C8 and, B3B11 compete with MHM6 top panel,FIG. 2).

FIG. 3 compares the in vitro IgE inhibitory activity of a particularprimate anti-human CD23 monoclonal antibody 5E8 to four differentPRIMATIZED® versions of said primate monoclonal antibody, the sequencesof which are described below.

-   -   p5E8G4P− This PRIMATIZED® antibody contains the following        sequences:        -   Human kappa light chain constant region and a human gamma 4            constant region which contains a P mutation (Angal et al.,            Mol. Immunol., 30:105-108 (1993));    -   p5E8G4PN− This PRIMATIZED® antibody contains the human kappa        light chain constant region and a human gamma 4 constant region        having a P mutation (Angal et al. Mol. Immunol., 30:105-108        (1993)). This antibody also contains a mutation in the heavy        chain variable region which changes an asparagine residue        (potential carbohydrate attachment site) to a lysine;    -   p5E8G1 This PRIMATIZED® antibody contains the human kappa light        chain constant region and a human gamma 1 constant region;    -   p5E8G1N− This PRIMATIZED® antibody contains the human kappa        light chain constant region and human gamma 1 constant region.        This antibody also contains a mutation in the heavy chain        variable region which changes an asparagine residue        (carbohydrate attachment site) to a lysine;

FIG. 4 contains a table which compares the apparent Kd in nM of theantibodies identified in FIG. 3 and summarizes their IgE suppressiveactivity.

FIG. 5 compares the in vitro IgE inhibitory activity of a particularprimate anti-human CD23 monoclonal antibody, 6G5, to two differentPRIMATIZED® versions of 6G5 which are described below:

-   -   p6G5G1 This PRIMATIZED® antibody contains the human lambda light        chain constant region and the human gamma 1 constant region;    -   p6G5G4P This PRIMATIZED® antibody contains the human lamda light        chain constant region and the human gamma 4 constant region with        a P mutation (Angal et al., Mol. Immunol., 30:105-108 (1993));

FIG. 6 compares the in vitro IgE inhibitory activity of primateanti-human CD23 monoclonal antibody 2C8 to F(ab′)₂ derived from 2C8;

FIG. 7 shows that the F(ab′)₂ derived from 2C8 antagonizes thesuppression of in vitro IgE activity of primate anti-human CD23monoclonal antibody 2C8.

FIG. 8 shows the in vivo IgE inhibitory activity of a particular primateanti-human CD23 monoclonal antibody, 5E8, in a SCID animal model;

FIG. 9 compares the in vivo inhibitory activity of primate anti-human6G5 and a PRIMATIZED® version thereof p6GSG4P.

FIG. 10 shows the in vivo IgE inhibitory activity of the primateanti-human CD23 monoclonal antibody 6G5 and a PRIMATIZED® versionthereof, p6G5G1.

DEFINITION OF TERMS USED IN THIS APPLICATION Chimeric Antibody:

A recombinant antibody containing regions from two different antibodies,usually different species antibodies, most typically rodent variablesequences and human constant domain sequences.

Anti-Human CD23 Gamma 1 Antibody

An antibody that specifically binds human CD23 which contains a humangamma 1 constant region or fragment or modification thereof whichinhibits induced IgE production. This includes, in particular,antibodies containing rodent or primate variable domains or antigenbinding portions, humanized, PRIMATIZED®, and human anti-human CD23monoclonal antibodies which comprise a human gamma 1 constant domain,fragment, or modification thereof, which inhibit induced IgE productionin vitro.

Anti-Human CD23 Gamma 3 Antibody

An antibody that specifically binds human CD23 which contains a humangamma 3 constant region or fragment or modification thereof whichinhibits induced IgE production. This includes, in particular,antibodies containing rodent or primate variable domains or antigenbinding portions, humanized, PRIMATIZED®, and human anti-human CD23monoclonal antibodies which comprise a human gamma 3 constant domain,fragment, or modification thereof, which inhibit induced IgE productionin vitro.

Modifications of Antibody Constant Domains

Antibodies according to the present invention containing mutations,substitutions or deletions of the constant region, that may create adesired change in the level of efficiency, i.e. in FcR binding, withoutchanging the basic effector functions mediated by the constant region.

PRIMATIZED® Antibody

A recombinant antibody containing primate variable sequences or antigenbinding portions, and human constant domain sequences.

Humanized Antibody:

A recombinant antibody containing a non-human variable region or antigenbinding portion which has been modified to more closely mimic a humanantibody variable region and thereby eliminate or minimize potentialimmunogenicity if administered to humans without sacrificing thespecificity or affinity of the immunoglobulin. There are several knownmethods of humanization, including, “veneering” which comprises selectmodification of surface residues, framework replacement, (CDR grafting)and molecular modeling.

Gamma 1 Constant Domain:

A particular type of constant domain sequence which confers upon anantibody specific effector activities. In the present application, gamma1 constant domain refers to a human gamma 1 constant domain, fragment ormodification thereof, which retains gamma 1 effector functions incombination with anti-CD23 variable domain sequences or antigen bindingportions. Modifications include human gamma-1 constant domains whichcomprise the deletion, substitution or addition of one or more aminoacid residues. This effector function is manifested by the ability of anantibody containing such a constant domain to inhibit induced IgEproduction.

Gamma 3 Constant Domain:

A particular type of constant domain sequence which confers upon anantibody specific effector activities. In the present application, gamma3 constant domain refers to a human gamma 3 constant domain, fragment ormodification thereof, which retains gamma 3 effector functions incombination with anti-CD23 variable domain sequences or antigen bindingportions. Modifications include human gamma-3 constant domains whichcomprise the deletion, substitution or addition of one or more aminoacid residues. This effector function is manifested by the ability of anantibody containing such a constant domain to inhibit induced IgEproduction.

CD23:

This refers to the low affinity receptor for IgE, FceRII/CD23.

Anti-CD23 Antibody:

An antibody that specifically binds CD23, preferably human CD23.

DETAILED DESCRIPTION OF THE INVENTION

As discussed supra, while many groups have previously reported theproduction of anti-CD23 antibodies and the use thereof as antagonistsand agonists for modulating IgE production, the exact mechanism by whichsuch antibodies modulate IgE expression in systems where IL-4 inducesIgE production remains unclear. Thus, it would be beneficial if themeans by which such antibodies modulate IgE expression were elucidated,or at least better explained, as such information would be potentiallyuseful in designing therapeutics for treatment of diseases whereinmodulation of IgE production is therapeutically desirable. Inparticular, it would be beneficial if improved antibodies specific toCD23 were obtained having improved capacity to inhibit induced IgEproduction, as enhanced IgE levels are believed to be involved innumerous disease processes, e.g., allergic conditions, inflammatoryconditions and autoimmune diseases. Such diseases include by way ofexample, atopic dermatitis, eczema, allergic rhinitis and conjuntivitis,Job's syndrome, and asthma.

Toward that end, the present inventors have surprisingly discovered thatanti-human CD23 monoclonal antibodies which contain human gamma-1constant domains inhibit IgE production in systems where IgE productionis induced by IL-4 significantly better than CD23 monoclonal antibodiesof other effector types, e.g., those comprising human gamma-4 constantdomains or CD23 monoclonal antibodies or antibody fragments lackingeffector functions altogether. Because human gamma-3 constant domainshave been shown to mediate the same effector functions as human gamma-1,anti-human CD23 monoclonal antibodies containing human gamma-3 constantdomains are also included.

There are currently five defined effector functions for the IgG (gamma)class of antibodies. Two of these functions, complement activation andFcγRN interaction, are not found in the in vitro assays described in thepresent invention, and are therefore not likely to be involved in themolecular mechanism. Three other FcγR receptors have been identifiedwhich interact with the IgG class of antibodies: FcγRI, FcγRII (of whichthere are at least six different proteins) and FcγRIII (with at leasttwo different proteins). All three of these receptors interact with bothIgG1 and IgG3.

FcγRI is the only one of the three having an appreciable affinity forIgG. It binds both monomeric gamma-1 and gamma-3 with a K_(a) of about5×10⁸ M⁻¹. However, its affinity for human gamma-4 is about 10-foldless, and it does not bind human gamma-2 at all (Fries et al., 1982, J.Immunol. 129: 1041-1049; Kurlander and Batker, 1982, J. Clin. Invest.69: 1-8; Woof, 1986, G. Mol. Immunol. 21: 523-527; see also Burton andWoof, 1992, Human Antibody Effector Function, Adv. Immunol. 51: 1-84).

Although the affinity of human FcγRII and FcγRIII for human IgG isgenerally very low (K_(a)<10⁷ M⁻¹), affinity for human IgG1 and humanIgG3 increases significantly (K_(a)=2 to 5×10⁷ M⁻¹) when they are boundto antigen (Karas et al.; 1982, Blood 60: 1277-1282). The affinity ofhuman FcγRII for human IgG2 bound to antigen has given conflictingresults. Human FcγRIII does not bind to human IgG2. Human FcγRII andhuman FcγRIII do not bind to human IgG4 (Van de Winkel and Anderson,1991, J. Leuk. Biol. 49: 511-524; Huizing a et al., 1989, J. Immunol.142: 2359-2364).

While Fc mediated effector functions are sometimes significant to thetherapeutic activity of antibodies, this discovery was surprising in thecase of anti-CD23 antibodies because the role of effector function inthe IgE inhibitory activity of anti-CD23 antibodies had not beenpreviously reported. In fact, previous evidence had suggested thatantibody effector function was not significant to the ability ofanti-CD23 antibodies to inhibit induced IgE production. For example,Flores-Romo et al., Science, 261:1038-1041 (1993) had reported that Fabsprepared from a polyclonal anti-CD23 antibody inhibited an in vivoinduced IgE antigen-specific response.

The discovery that effector functions mediated through the constantregion of the anti-CD23 antibodies are apparently involved was madeafter the present inventors isolated various primate antibodies specificto CD23 having anti-IgE inhibiting activity and compared theseantibodies to PRIMATIZED® versions with respect to their ability toinhibit IL-4 induced IgE production in vitro and in vivo. Antibodiesconstructed with a human gamma-4 constant region failed to inhibit IgEantigen-specific responses in vitro, whereas antibodies containing ahuman gamma-1 constant region were successful.

Because one (or more) of the three classes of FcγR receptors, FcγRI,FcγRII or FcγRIII, is likely involved in the specific effector functionmediated through the gamma-1 domain, and because these classes ofreceptors also bind to antibodies containing gamma-3 domains, it islogical that anti-CD23 antibodies containing gamma-3 domains will alsobe successful at inhibiting IgE-antigen specific-response in vitro.

More specifically, and as described in greater detail infra, fiveprimate monoclonal antibodies which specifically bound both cellular andsoluble CD23 were isolated from an Old World monkey (macaque) accordingto the methodology which is disclosed in commonly assigned applicationSer. No. 08/379,072 (now allowed), which application is incorporated byreference in its entirety herein. This application described in detail ameans for producing monoclonal antibodies to desired antigens, desirablehuman antigens, in Old World monkeys and their advantages in relation toantibodies of other species as therapeutics, for example reduced orpotentially lack of immunogenicity in humans because of the phylogeneticcloseness of humans and Old World monkeys. In fact, because of thephylogenetic closeness of these species, it is difficult to distinguishOld World monkey immunoglobulins from human immunoglobulins by sequencecomparison.

Four of these five primate monoclonal anti-human CD23 antibodies weredemonstrated to be capable of inhibiting IL-4 induced IgE production inan in vitro B cell assay described in detail infra and the most potentwas also shown to inhibit IL-4 induced IgE in a SCID mouse animal model(also described in detail infra). Based on this IgE inhibitory activity,and expected low immunogenicity in humans, such antibodies arepotentially suitable as therapeutics for treating diseases whereininhibition of IgE production is therapeutically desirable.

However, in order to further reduce immunogenicity, it was elected toPRIMATIZE® two primate monoclonal antibodies (a type of chimerization ofantibodies) according to the methodology which is also described in U.S.Ser. No. 08/379,072 (now allowed), incorporated by reference herein.PRIMATIZATION® essentially refers to the production of recombinantantibodies developed by IDEC Pharmaceuticals Corporation which compriseprimate variable regions and human constant regions. Primatization ofthe two primate anti-human CD23 monoclonal (5E8 and 6G5) antibodieshaving potent IgE inhibiting activity was effected in order to eliminateany potential immunogenicity attributable to the primate constantdomains in humans.

Again, because of the inventors' initial expectation from publishedliterature that Fc effector function was not necessary for induced IgEinhibition, human gamma 4 versions of these particular antibodies wereinitially produced. However, quite surprisingly, it was found that thegamma-4 versions produced from both of these primate monoclonalantibodies were ineffective, i.e., they required significantly higherconcentrations of PRIMATIZED® gamma 4 antibody than the primate antibodyto inhibit IL-4 induced IgE production in in vitro assays.

Moreover, even more surprising was the discovery that when the same twoprimate antibodies were then converted to human gamma-1 versions (bysubstitution of the primate constant domains with human gamma-1 constantdomains), that these gamma-1 antibodies very effectively inhibitedinduced IgE production in vitro. Thus, our results suggested that Fceffector function is apparently significant to the ability of anti-humanCD23 antibodies to inhibit induced IgE production. This hypothesis wasconfirmed when a third primate anti-human CD23 monoclonal, i.e., the 2C8antibody, which was shown by us to inhibit IgE production in vitro, wasconverted to a F(ab′)₂, which was found to be substantially incapable ofinhibiting induced IgE production in vitro. In fact, this F(ab′)₂ wasfound to antagonize the suppressive effects on induced IgE blockingactivity of the primate anti-human CD23 monoclonal antibody 2C8.

In addition, it was found that removing a glycosylation site in theheavy chain variable region of one of the antibodies (5E8) had no effecton binding of the antibody to CD23 (as evidenced by obtained Kd values),or on induced IgE inhibition. Thus, the differences in IgE inhibitionwere shown to apparently not involve glycosylation differences.

The PRIMATIZED® gamma 1 version of primate 6G5 was found to inhibitinduced IgE expression in SCID mice while the same concentration ofeither the primate 6G5 or the PRIMATIZED® p6G5F4p did not inhibitinduced IgE expression. Therefore, an antibody containing human gamma-1constant domains was found to be even more effective in an in vivoanimal model then the primate monoclonal antibody. Furthermore, theinventors anticipate that anti-CD23 antibodies containing human gamma-3constant domains will be just as effective as those having gamma-1constant domains, because gamma-1 and gamma-3 constant domains haveaffinity for the same classes of Fc receptors.

Accordingly, based on these results, it has been surprisingly discoveredthat an active Fc region, in particular that of human gamma-1 or humangamma-3, is significantly involved in the mechanism of IL-4 induced IgEinhibition by anti-human CD23 monoclonal antibodies. This discovery isquite unexpected especially based on earlier reports that Fabs derivedfrom polyclonal anti-CD23 antibodies were capable of inhibiting inducedIgE production, and also based on the various theories as to how CD23affects induced IgE expression.

Accordingly, the present invention relates to anti-human CD23 antibodiescontaining human gamma-1 or gamma-3 constant domains and their use astherapeutics based on their ability to effectively inhibit IgEexpression.

The skilled artisan can prepare anti-human CD23 antibodies containingeither human gamma-1 or gamma-3 constant domains by methods which arewell known in the art for the manufacture of chimeric antibodies.Essentially, such methods comprise producing anti-human CD23 antibodiesin a desired host or in vitro, cloning a hybridoma or cell line whichproduces an anti-human CD23 monoclonal antibody exhibiting desirablecharacteristics, e.g., adequate CD23 binding affinity, cloning thenucleic acid sequences which encode such antibody from said hybridoma orcell line, e.g. by polymerase chain reaction using suitable primers,isolating the variable domains contained therein, recombining suchvariable domains with human gamma-1 or gamma-3 constant domains and theappropriate human light chain constant domain, and expressing theresultant nucleic acid sequence encoding a chimeric anti-human CD23gamma-1 or gamma-3 immunoglobulin in a suitable expression system.Preferably, the anti-human CD23 antibodies of the invention will haveapparent CD23 binding affinities ranging from 0.1 nM to 1000 nM, morepreferably at least 50 nM, and most preferably at least 5 nM.

Host cells suitable for expression of recombinant immunoglobulins arewell known in the art. For example, recombinant antibodies may beexpressed in Chinese hamster ovary (CHO) cells, DG44 or DUXB11; or CHOcells CHO K-1; mouse myeloma cells SP2/0 or X63-Ag8.653 or NSO; ratmyeloma cells YB2/0; baby hamster kidney cells, BHK; human embryonickidney line, 293; monkey kidney cells, CV1; human lung fibroblasts,WI38; human cervical carcinoma cells, HELA; insect cells, plant cells,yeast or in bacteria. Further, vectors suitable for expression ofimmunoglobulins are also well known in the art and are commerciallyavailable.

A particularly preferred vector system is the translationally impairedvector system disclosed in U.S. Ser. No. 08/147,696 (now allowed), whichcomprises a translationally impaired dominant selectable marker (neo)containing an intron into which a desired heterologous DNA is inserted.This vector system has been found to provide for very high yields ofrecombinant proteins, e.g., immunoglobulins. However, the subjectanti-CD23 antibodies may be produced in any vector system which issuitable for expression of functional immunoglobulins.

Also, the present invention embraces human monoclonal antibodies of thegamma-1 or gamma-3 types which are specific to human CD23. Methods forisolation of human monoclonal antibodies are also well known in the artand include in vitro methods, e.g., in vitro immunization of human Bcells in tissue culture, and in vivo methods, e.g. synthesis of humanmonoclonal antibodies in SCID mice. A preferred means of producing humanmonoclonal antibodies in SCID mice which combines in vitro priming ofhuman spleen cells which are then introduced into SCID mice is disclosedin U.S. Ser. No. 08/488,376 (incorporated by reference in its entiretyherein). This method is advantageous as it provides for the reproduciblerecovery of monoclonal antibodies having high affinity against a desiredantigen, e.g., a human antigen.

Also, the present invention embraces human monoclonal antibodies whichcompete with the primate anti-human CD23 monoclonal antibodies 5E8 and6G5 for binding to CD23.

EXAMPLE 1 Production of Primate Anti-CD23 Antibodies

Five primate monoclonal antibodies specific to CD23 were isolated frommacaques substantially according to the methodology disclosed in Ser.No. 08/379,072, which has been incorporated by reference herein. Theexact techniques utilized are described in detail below.

Methodology for Isolation and Characterization of Anti-Human CD23Monoclonal Antibodies

Purification of the Immunogen sCD23 from 8866 Cells

During purification, soluble CD23(sCD23) was quantified by a three-stepELISA using a murine anti-CD23 antibody (Binding Site; catalog #MC112)as a capture. The antigen was partially purified from cultures of 8866cells maintained in suspension bioreactors using RPMI 1640 (JRHBiosciences; catalog #56-509) supplemented with 10% fetal bovine serum(JRH Biosciences) and 4 mM glutamine (JRH Biosciences; catalog #90114)at 37° C. Carbon dioxide was used to maintain pH 7.1. After removingcells by 0.45 μm filtration, phenylmethyl sulfonyl fluoride (finalconcentration 0.2 mM, Sigman Chemical Co.; catalog #P-7626) andethylenediaminetetraacetic acid (final concentration 3 mM, SigmaChemical Co.; catalog #EDS) were added to the supernate and the solutionstored at 2-8° C. The cell-free supernate was concentrated approximately15 to 20-fold using a hollow-fiber ultrafiltration cartridge (A/TTechnology; catalog #UFP-10-C-9A; 10,000 d MWCO) or tangential flowultrafiltration cartridge (Filtron Corporation; 10,000 d MWCO) atambient temperature. The concentrated supernate was sterile filtered andstored at −70° C. Thawed concentrates were de-lipidated by adding SM-2BioBeads (BioRad Industries; catalog #152-3920) at 5 g/L and stirringovernight at 2-8° C. The resin was removed by filtration and thesolution stored at 2-8° C. For some preparations of sCD23, concentrateswere fractionated using ammonium sulfate (35-70% (w/v); Fisher; catalog#A702-3) before or after de-lipidation.

The de-lipidated solution was subsequently purified using affinitychromatography at 2-8° C. The affinity matrix was prepared by covalentlylinking a murine anti-CD23 monoclonal antibody (BU38) to Sepharose usingCNBr-activated Sepharose 4B (Sigma Chemical Co.; catalog #C-9142). TheBU38 antibody was purified to >90% homogeneity from ascites (BindingSite; catalog #CUS830) using Protein A chromatography. The de-lipidatedsolution was applied to the affinity column (1.5×5 cm) equilibrated with1×PBS (Gibco BRL; catalog #70013-0.32), pH 7.2 and the column washedwith 1×PBS, pH 7.2, containing 0.05% NP40 (Sigma Chemical Co.) to removenon-bound protein. Soluble CD23 was eluted using 3.5 M MgCl₂ (Fisher;catalog #M33-500). Fractions containing sCD23 were combined and dialyzed(Baxter Spectra/Por; catalog #D1615-1) against 1×PBS, pH 7.2 at 2-8° C.After dialysis, the protein solution was concentrated by centrifugationusing Centriprep 10 spin filters (Amicon Corporation; MWCO 10,000 d) andpreparations stored at −70° C. The purity of sCD23 was estimated tobe >70% using SDS-PAGE analysis (4-20% pre-cast gels, Novex Corporation)and Coomasie staining.

Immunization of Primates and Isolation of Immune Cells

Cynomolgus monkeys (White Sands Research Center, Alamogordo, N. Mex.)were immunized with soluble CD23 which had been purified from thesupernatant of human RPMI 8866 cells (B cell lymphoma, Hassner andSaxon, J. Immunol., 132:2844 (1984)). Each monkey was immunized everythird week with 200 μg soluble CD23 in 500 μl PBS mixed with 167 μlTemuritide (adjuvant peptide) (Sigma, St. Louis, Mo., Catalog #A-9519)and 333 μl 3× PROVAX® (IDEC Pharmaceuticals Corporation). Immunizationwas effected intradermally, intraperitoneally, intramuscularly andsubcutaneously. The titer of anti-CD23 antibodies in the serum of themonkeys was measured by ELISA on 8866 cells and compared to a pre-bleedfrom the same monkeys.

Monkey PRO978, with a serum titer of fifty thousand was sacrificed, andthe spleen and lymph nodes were surgically removed, and shipped on iceto IDEC pharmaceuticals, submerged in sterile RPMI-1640 (Gibco BRL,Gaithersburg, Md., Catalog #21870-050) supplemented with 10% fetal calfserum, 2 mM L-glutamine, 2 mM sodium pyruvate and 50 μg/ml gentamicin.Immediately upon arrival the spleen was homogenized by squeezing itthrough a wire mesh with a glass pistil. Red blood cells were lysed inan ammonium chloride based hypotonic buffer and the remaininglymphocytes collected and washed in RPMI-1640 at least three times.Lymph nodes were homogenized similarly into a single cell suspension,collected and washed at least three times in RPMI-1640.

Production of Hybridomas

After the last wash, the cells were counted, and the primate cellsobtained above were then somatically fused to the mouse-humanheterohybridoma cell line H6K6/B5 (Carroll et al., J. Immunol. Methods,89:61 (1986)) using standard techniques (Boerner et al., J. Immunol.,147:86) (1991)) and plated into 96 well dishes (175 dishes or 14,700wells for the spleen, and 17 dishes or 1386 wells for the lymph nodes)at 300,000 cells per well.

This procedure involved the mixing of lymphocytes and theabove-identified fusion partner, at a 2:1 ratio, which cells were slowlyresuspended into 50% PEG 1500 (Sigma, Catalog #P5402) for 1 minute.These cells were then allowed to rest for 1 minute and then slowlyfurther resuspended in excess RPMI-1640. Afterward, the cells were againallowed to rest, this time for 15 minutes before a light spin at 250×9.The cells were then resuspended in RMPI-1640 growth media, which wassupplemented with 20% Fetal Calf Serum, 2 mM L-Glutamine, SodiumPyruvate, Non-Essential Amino Acids and 50 μg/ml Gentamicin, containing100 μM Hypoxanthine, 16 μM Thymidine (BoehringerMannheim, Germany, #623091) and 5.8 μM Azaserine (Sigma, Catalog #A 1164) (HTA). HTA is aselection agent which provides the survival of successfully fused cells(primate lymphocyte fused with heterohybridoma fusion partner).

Approximately 65% of the wells showed growth (10,500 wells). These wellswere then screened for the presence of anti-human CD23 antibody by athree step cell ELISA.

ELISA Procedure

The first step of the ELISA comprised the transferral of fiftymicroliters of supernatant from each well to ninety-six well plateswhich had previously been coated with 10⁵ 8866 cells (CD23 positive cellline) per well. These plates were made by first coating the plates with50 μl of aqueous solution containing twenty μg/ml Poly L-Lysine (SigmaCatalog #P1399, MW 150,000-300,000) for thirty minutes at roomtemperature. The remaining solution was removed (“flicked out”) and theplates left to dry. Once dry, fifty μl of 8866 cells in PBS weretransferred and spun at 600 g for five minutes. The 8866 cells werecovalently bound to the plate by adding fifty μl 0.5% glutaraldehyde(Sigma Catalog #G6257) in phosphate buffered saline (PBS) for 15minutes. The glutaraldehyde was removed (flicked out) and the platesblocked with one hundred fifty μl 100 mM glycine (Sigma Catalog #G-2879)in 0.1% BSA-PBS. After the addition of supernatants, the plates wereincubated at 37° C. for one to two hours and washed seven to nine timeswith tap water, and a goat anti-human IgG antibody coupled to horseradish peroxidase (HRPO) (Southern Biotech, Birmingham, Ala., Catalog#2040-05) diluted 1:2000 into 1% dry skimmed milk (Vons) in PBS—0.05%Tween 20 (Sigma, Catalog #P1379) was added. The plates were incubatedfor forty-five minutes at 37° C., and again washed seven to nine timesin tap water. The presence of the HRPO was detected by a colordevelopment after the addition of a TMB reagent (Kirkegaard & Perry,Gaithersburg, Md., Catalog #50-76-02 and 50-65-02), 100 μl/well. Thereaction was stopped by adding twenty-five μl 4NH₂SO₄. Optical density(OD) was measured at 470 nM on a spectrophotometer (Titertek Multiscan).The OD values greater than two times the background were scored aspositive.

The second step in the ELISA was effected to confirm that thesupernatants which had been scored positive in the first ELISA reactedto CD23 and not to some irrelevant antigen. This was effected by testingthe supernatants on SupT1 cells (ERC BioServices Corporation, Rockville,Md., Catalog #100), a CD23 negative human cell line, using the sameELISA procedure. Supernatants that scored similarly in both tests werediscarded. These results indicated that fifty-six of the 10,500 wellswith growth showed the presence of a primate monoclonal antibody thatbound to 8866 cells in two separate screenings at different times anddid not bind to SupT1 cells.

The third step of the ELISA was conducted to determine whether thesupernatants identified according to the first two ELISA steps, reactedwith soluble CD23. In this third ELISA, 96 well plates were coated at 4°C. overnight with 2 μg/ml BG-6 (Biosource International, Camarillo,Calif., Catalog #CT-CD23-CF), a mouse monoclonal antibody that binds tosoluble CD23 but does not block CD23-IgE binding, contained in a 50 mMbicarbonate buffer, pH 9.3. After removing the coating buffer, fifty μlof semi-purified soluble CD23 at a predetermined dilution in PBS wereadded to the plate and incubated for two hours at room temperature.After washing the plate with tap water seven to nine times, 50 μlsupernatants from selected wells were added. After washing the plate intap water seven to nine times, 50 μl rabbit anti-human IgG (mouseadsorbed)-HRPO (Southern Biotech, Catalog #6145-05) diluted 1:4000 in 1%dry skimmed milk in PBS with 0.05% Tween 20 were added, incubated fortwo hours at 37° C., washed seven to nine times in tap water anddeveloped with TMB as described above. Wells with OD's greater than twotimes the background were again scored as positive.

Twenty-one of the fifty-six wells that showed binding to 8866 cells alsobound to sCD23 in the ELISA. These wells were expanded and subcloned atleast twice by plating out cells at one cell per three wells. Afterapproximately three months, five stable hybridomas producing primatemonoclonal antibodies to CD23 were obtained.

Antibody Purification by Protein A Methods

Essentially, antibodies are purified by centrifugation of the culturesupernatant to remove cells and debris. The resultant centrifugedsamples are then filtered through a 0.2 μm filter. A protein A sepharoseFast flow column is then prepared and equilibrated using PBS (pH 7.4).The supernatant is then loaded on the column at an appropriate flow rate(2 ml/min). After loading, the wash column is washed with 10 columnvolume of PBS (pH 7.4). The antibody is then eluted from the column withelution buffer (0.2 M acetic acid, 0.1 M glycine pH 3.5) at 1 ml/minflow rate. One milliliter fractions/tube (2.0 M Tris-Hcl pH 10.0)including 100 μl of Tris, are then collected. Afterward,spectrophotometer readings are taken at 280 nm. The resultant fractionswith high absorbance at 280 nm containing the antibody are thencollected and dialyzed against PBS overnight. The product is thensterilized by filtration through 0.22 μm membrane and stored at −20° C.

Four of these five primate anti-human CD23 monoclonal antibodies (1H6,2C8, 5E8 and 6G5) were demonstrated to inhibit IgE production in an invitro assay which measures IgE production by IL4-hydrocortisone inducedperipheral blood mononuclear cell (PBMC) cultures. These results areshown in FIG. 1. The assay conditions are described below. The fifthprimate monoclonal anti-human CD23 antibody B3B11 was inactive in thisassay.

IL-4 Stimulated IgE Production by Peripheral Blood Mononuclear Cells

As discussed supra, the subject primate antibodies and PRIMATIZED® formsthereof were assessed for their ability to inhibit IgE production in anin vitro assay which measured the effect of such antibodies on IgEproduction by IL-4 stimulated peripheral blood mononuclear cells.

Materials for In Vitro IL-4 IgE Assay

Forty-eight well flat bottom cluster plates (Costar Catalog #3548) (1.5million PBMCs per ml per well (48 well plate))

Human recombinant IL-4 (Genzyme Catalog #2181-01; 10 μg (2.5×10⁷ units)

anti-CD23 Mabs:

-   -   murine Mab (MHM6; DAKO. Catalog #M763)    -   primate Mabs (no preservatives)    -   PRIMATIZED® (no preservatives)

HB101 basal medium: (Irvine Scientific Catalog #T000)

HB101 supplement: (Irvine Scientific Catalog #T151)

Fetal Bovine Serum: (FBS; Bio-Whittaker Catalog #14-501F)

dimethylsulfoxide: (DMSO; Fisher Scientific Catalog #D128-500)

hydrocortisone: (Sigma Catalog #H-0888)

puromycin: (Sigma Catalog #P-7255)

cyclohexamide: (Sigma Catalog #C-7698)

Histopaque®: (Sigma Catalog #H-8889)

Hank's Buffered Salt Solution: (HBSS; Irvine Scientific Catalog #9232)

1% FBS in HBSS

concentrated Dulbeccols phosphate buffered saline (10×DPBS;Bio-Whittaker, Catalog #17-517Q)

Bath Clear Microbicide (Fisher, Catalog #13-641-334) in DPBS

Solutions:

puromycin solution: 40 μg/ml in HB101 growth medium cyclohexamidesolution: 200 μg/ml in HB101 growth medium hydrocortisone solution: 0.1M solution in DMSO anti-CD23 murine Mabs were extensively dialyzed toremove preservatives HB101 growth medium HB101 basal medium 500 ml HB101supplement in 10 ml 5 ml sterile filtered distilled H₂O FBS 10 mlhydrocortisone solution 0.25 ml (final conc. 5 μM)

In Vitro Assay Procedure

Buffy coat cells 1:4 are diluted using HBSS at room temperature. Thesecells are derived from whole blood after an overnight incubation at roomtemperature to resolve and separate the plasma components, clottedplatelets and fibrin, and buffy coat cells.

Thirty microliters of diluted buffy coat are then overlayed onto fifteenmicroliters of Histopaque in fifty ml conical tubes. These tubes arethen centrifuged for twenty minutes at 1700 rpm at room temperaturewithout brakes (IEC 216 swinging bucket rotor). The white PBMC layer isthen collected using a sterile pipette, taking care not to disturb theother layers. The PBMCs (peripheral blood mononuclear cells) are thebuffy coat cells which have been sedimented by centrifugation partiallythrough a HISTOPAQUE® density gradient to form a distinctly visiblewhite layer of cells. These cells are collected with a pipette, rinsedwith HBSS, and then counted using a hemocytometer. Typically, 300 to 600million PBMCs can be recovered from a single 450 ml buffy coat package.

The collected PBMCs are then washed three times in 1% FBS/HBSS. Thewashed cells are collected by centrifugation for seven minutes at 1300rpm at 7° C.

The number of cells collected is then determined using a hemocytometer.The cell concentration is adjusted to about three million cells permilliliter of HB101 growth medium.

Approximately about 1.5 million cells (0.5 ml) are then added to eachwell of a 48 well plate. In general, five replicate samples are preparedfor each experiment. The perimeter wells of each plate are not used forcell samples. Accordingly, these wells are filled, e.g., using 0.5 ml of0.05% BathClear/DPBS.

0.5 ml HB101 growth medium containing desired amounts of IL-4 and Mab isthen added to the wells. The IL-4 used is recombinant DNA-generatedhuman interleukin 4. The Mab used in the assay is a murine, primate orPRIMATIZED® antibody. Typically, IL-4 is added at a final concentrate of100 U/ml and Mab is added at a final concentrate ranging from 0.01 to 3μg/ml.

The cells are then incubated for nine to eleven days at 37° C. in amoist incubator set at 5% CO₂. After incubation, the supernatant fluidsare collected and the IgE content is measured.

IgE ELISA

The following list identifies materials and solutions used in the IgEELISAs.

Materials and Solutions Needed for IgE ELISA

sulfuric acid, 4 Mcoating buffer: 10 mM sodium bicarbonate buffer, pH 9.6 concentratedphosphate buffered saline (10×PBS) stock solution:

NaH₂PO₄ 26.6 gm Na₂HPO₄ 289 gm NaCl 1064 gm distilled H₂O 10 Lblocking buffer: 10% FBS/PBSdilution buffer: 1% BSA/0.05% Tween 20/PBSwashing buffer: 0.05% Tween 20/PBSgoat anti-human IgE (epsilon chain-specific), unlabeled: (Tago Catalog#4104)human IgE standard: (The Binding Site Catalog #BP094)goat anti-human IgE, HRP-labeled: (Tago Catalog #AHI 0504)TMB peroxidase substrate: (KPL Catalog #50-76-02)peroxidase solution B: (KPL Catalog #50-65-02)working substrate solution: mix substrate and Solution B at 1:1 ratioImmulon II microtiter plates (Dynatech Labs Catalog #011-010-3455)

IgE ELISA Procedure

Each well of a microtiter plate is coated using 100 μl of a coatingbuffer containing 2 μg/ml goat anti-human IgE.

The coated plate is then incubated overnight at 4° C.

After incubation, each well in the plate is then washed three times with200 μl of Tween 20/PBS. After washing, the non-specific binding sitesare blocked with 200 μl blocking buffer/well for 1 hour at 37° C.

One hundred μl of samples or standards are then added to each well;which wells are then incubated overnight at 4° C. After incubation, thesamples are tested with or without dilution. A standard concentrationcurve is prepared for each plate using several dilutions of IgE rangingfrom 0.1 to 50 ng/ml.

After overnight incubation, each plate is washed five times with Tween20/PBS.

One hundred μl of horseradish peroxidase (HRP) labeled goat anti-humanIgE diluted 1:10,000 in dilution buffer is then added to each drainedwell. The plate is then incubated for 4 hours at 37° C.

The plates are then washed 5 times with Tween 20/PBS and 3 times withwater.

One hundred μl of 3,3′,5,5′-tetramethylbenzidine working substratesolution is then added to each well. The plate is then incubated fortwenty-five minutes in the dark at room temperature. After incubationthe developing reaction is stopped by the addition of fifty μl of 4 Msulfuric acid.

The absorbency is then read concurrently at 450 and 540 nm. The 540 nmabsorbency values are subtracted as background.

Assay for Kd Measurement of Primate Monoclonal Anti-Human CD23Antibodies Scatchard Analysis Procedure 1. Radiolabeling Procedure

IODO-BEADS are washed with 10 mN Phosphate Buffer, pH 7.4 twice using 1mL of buffer per 2 beads. The beads are then dried on filter paper.

The two beads are then added to 100 μl ¹²⁵I solution, containing about 1mCi of I, diluted with 200 μl of the phosphate buffer, and left at roomtemperature for 5 minutes.

The antibody (50 μgs) is added to the preloaded beads. The reaction timefor maximal incorporation of radioactivity is 6 minutes.

The reaction is stopped by removing the radiolabeled antibody from thereaction vessel.

Gel filtration is then performed to remove excess ¹²⁵I or unincorporated¹²⁵I from the radiolabeled antibody solution. This is effected bypassing the radiolabeled antibody over a column made up of 1.5 mLSephadex-G25, 1.5 mL DEAE Sephadex-A25 and 0.5 mL Amberlite. Theradiolabeled antibody is eluted off in a total volume of 5 mL at aconcentration of about 10 μg/mL. (Elution Buffer: 1×PBS containing 10%Gelatin, 2% Sodium Azide and 1% BSA).

2. Optimization Assay (Direct Binding Study)

The specific activity of the 10 μg/mL radiolabeled solution isdetermined by taking a 1 μl sample and running the sample on a gammacounter.

Example

1 × 10⁵  cpm/µl × 1000  µl/10  µg  antibody1 × 0⁵  cpm/µg  antibody 1 × 0⁴  cpm/ng  antibodyMolecular  wt.  of  antibody = 75, 000  ng/nmole

-   -   Specific activity:    -   1×10⁴ cpm/ng×75,000 ng/nmole=7.5×0⁸ cpm/nmole

The antigen-coated plate is blocked (to eliminate non-specific binding,e.g., with mB7.1-CHO) and the background plate (i.e., Untransfected-CHO)for one hour at room temperature-with 200 μl/well of blocking buffer(Blocking Buffer: 1×PBS containing 10% Gelatin, 2% Sodium Azide, 1% BSAand 10% FBS).

The plate(s) are then washed, typically ten times by hand with tapwater.

The 10 μg/mL radiolabeled antibody (50 μls) is then titrated by two-foldserial dilutions across the plate(s) using a multichannel pipette.Incubate for one hour at room temperature.

The plate(s) are again washed about 6-7 times with 200 μl/well of washbuffer (Wash Buffer:—1×PBS containing 10% Gelatin and 2% Sodium Azide).

The radio activity counts in each well are then determined by runningthe wells on a gamma counter.

The optimal radiolabeled antibody concentration is the concentration inwhich the difference between the specific counts and background countsis at a maximum.

3. Scatchard Analysis of Competition Assay

The 10 μg/mL radiolabeled solution is diluted to the optimalconcentration determined in the Direct Binding experiment.

The antigen-coated plate and the background plate are blocked for onehour at room temperature with 200 μl/well of blocking buffer.

The plate(s) are then washed, e.g., about 10 times, by hand with tapwater.

The “cold” (no radiolabel) antibody is then titrated by two-folddilutions in a separate U-bottom microtitre plate. The startingconcentration of the “cold” antibody should be at least 100 timesgreater than that of the optimal radiolabeled antibody concentration.

Example

-   -   Optimal Radiolabeled Conc.: 0.5 μg/mL    -   “Cold” Antibody Conc.: 100 μg/mL (Note: 1:2 titration in the        first well will adjust the “cold” antibody concentration to 50        μg/mL.)

Fifty μl/well of optimal radiolabeled antibody are then added to thewells containing “cold” antibody.

One hundred μl/well of the mixed solution are then transferred to thecorresponding wells of the antigen-coated plate, and incubated for onehour at room temperature.

Also, it is desirable also that the following controls be effected:

a) Direct binding of radiolabeled antibody to antigen-coated plate (5wells),

b) Direct binding of radiolabeled antibody to background plate (5wells).

After incubation, the plate(s) are washed, e.g., about 6-7 times, with200 μl/well of wash buffer.

The radio activity counts in each well are then determined by runningthe wells on a gamma counter.

These calculations are determined by calculating the specific counts ineach well tested by subtracting the background counts from the countsbound to the antigen-coated plate.

4. Calculations for Scatchard Analysis

The Molar Concentration of Bound antibody [B] can then be determined asfollows:

Example: At 50 μg/mL “cold antibody”

  Specific  counts  bound:  4382  cpm${Counts}\mspace{14mu} {bound}\mspace{14mu} {in}\mspace{14mu} {the}\mspace{14mu} {presence}\mspace{14mu} {of}\mspace{14mu} 0\mspace{14mu} {µg}\text{/}{mL}\mspace{14mu} {``{cold}"}\mspace{14mu} {ab}\text{:}\mspace{11mu} 215\mspace{14mu} {cpm}$  Difference:  4382  cpm − 215  cpm = 4167  cpm  Specific  Activity  (radiolabeled  ab):5.54 × 10⁹  cpm/nmole  4167  cpm ÷ 5.54 × 10⁹  cpm/nmole = 7.52 × 10⁻⁷  nmole$\begin{matrix}{{7.53 \times 10^{- 7}\mspace{14mu} {{nmole} \div 0.05}\mspace{14mu} {{mL}\left( {{sample}\mspace{14mu} {{vol}.}} \right)}} = {1.50 \times 10^{- 5}\mspace{14mu} {nmole}\text{/}{mL}}} \\{= {1.50 \times 10^{- 8}\mspace{14mu} {µmole}\text{/}{mL}}} \\{\lbrack B\rbrack = {1.50 \times 10^{- 11}\mspace{14mu} {mole}\text{/}{mL}\mspace{11mu} (M)}}\end{matrix}$

Total Molar Concentration [T] is determined as follows:

$\begin{matrix}{{50\mspace{14mu} {µg}\text{/}{mL} \times 1\mspace{14mu} {µmole}\text{/}75,000\mspace{14mu} {µg}} = {6.67 \times 10^{- 4}\mspace{14mu} {µmole}\text{/}{mL}}} \\{= {6.67 \times 10^{- 7}\mspace{14mu} {mmole}\text{/}{mL}\mspace{14mu} (M)}} \\{\lbrack T\rbrack = {66667 \times 10^{- 11}\mspace{14mu} {mmole}\text{/}{mL}\mspace{14mu} (M)}}\end{matrix}$

Free antibody [F] is determined as follows:

$\begin{matrix}{{{Free}\mspace{14mu} {Molar}\mspace{14mu} {{Conc}.}} = {{Total}\mspace{14mu} {minus}\mspace{14mu} {Bound}}} \\{\lbrack F\rbrack = {\left( {66667 \times 10^{- 11}} \right) - \left( {1.50 \times 10^{- 11}} \right)}} \\{= {66665.5 \times 10^{- 11}\mspace{14mu} {mmole}\text{/}{mL}\mspace{14mu} (M)}}\end{matrix}$

Calculate B/F.

Plot B versus B/F on Cricket Graph software.

Activity and Affinity of Anti-Human CD23 Antibodies According to theInvention

Four of the five isolated primate anti-human CD23 monoclonal antibodies(B3B11, 2C8, 5E8 and 6G5) were found to inhibit IgE production in theabove-identified in vitro assay which measures IgE production byIL4-hydrocortisone induced peripheral blood mononuclear cell (PBMC)cultures. These results are shown in FIG. 1. The fifth primatemonoclonal anti-human CD23 antibody 3G12 was inactive in this assay.

Two of the four primate monoclonal anti-human CD23 antibodies (B3B11 and2C8) found to be active in this in vitro assay were found to competewith a commercially available mouse anti-human CD23 antibody MHM6 (CAKOA/S, Glostrup, Denmark Catalog #M763). (FIG. 2, top panel.) However, inrepeated assays these antibodies were not as potent IgE inhibitors asMHM6 (data not shown). By contrast, the other primate anti-CD23monoclonal antibodies (5E8 and 6G5) were found to compete with eachother and did not complete with MHM6. (FIG. 2, middle and bottompanels.) Moreover, the primate anti-human CD23 monoclonal antibody 5E8was found to be a potent inhibitor of IL-4 induced IgE in the in vitroassay. (See FIGS. 1 and 3.)

Modified Hu-SCID-Mouse Model for Human IgE Synthesis and Measuring theInhibition of IL-4 Induced IgE Production by Anti-CD23 Antibodies InVivo

A modified hu-PBMC-SCID mouse model was also developed to detect theeffect of the subject antibodies on induced human IgE production invivo. PBMCs obtained from two donors were cultured with IL-4 in vitrofor two days. PBMCs were pooled and used to reconstitute groups ofC.B.-17 SCID mice with and without antibodies. Mice were bled on day 14,21, 28 and 35 and serum IgG and IgE levels were determined by ELISA.This in vivo model was used to assay primate and two different versionsof PRIMATIZED® antibodies to CD23 for their ability to inhibit theproduction of IgE.

A modified SCID mouse model was used because it is known that severecombined immunodeficiency scid/scid (SCID) mice, C.B.-17 (Bosma et al.,Nature, 301:527 (1983)) reconstituted with human peripheral bloodmononuclear cells (hu-PBMC-SCID) can produce significant quantities ofhuman immunoglobulins (Ig) (Moiser et al., Nature, 335:256 (1988);Moiser et al., J. Clin. Immunol., 10:185 (1990); Abedi et al., J.Immunol., 22:823 (1992); and Mazingue et al., Eur. J. Immunol., 21:1763(1991).) The predominant isotype of human immunoglobulin (Ig) producedin hu-PBMC-SCID mice is IgG. Generally, IgM, IgA and IgE isotypes arefound in very low or non-detectable levels except in cases where PBMC isobtained from donors with certain autoimmune or allergic diseaseconditions. It has also been reported that manipulation of hu-PBMC SCIDmouse model with certain cytokines may be provided for the generation ofsignificant levels of non-IgG isotypes, including IgE (Kilchherr et al.,Cellular Immunology, 151:241 (1993); Spiegelberg et al., J. Clin.Investigation, 93:711 (1994); and Carballido et al., J. Immunol.,155:4162 (1995)). The hu-PBMC-SCIDs, has been also used to generateantigen specific Ig provided the donor has been primed for the antigenin vivo.

Therefore, the aim of the present inventors was focused on establishinga suitable human IgE producing hu-PBMC-SCID mouse model that could beused to test the efficacy of therapeutic for treatment of IgE relateddiseases such as allergic disorders, including the subject anti-CD23antibodies.

Materials and Methods:

The following materials and methods were used in the hu-PBMC-SCID mousemodel described below.

SCID mice: C.B-17 scid/scid immunodeficient mice were obtained fromTaconic (C.B.-17/IcrTac-scidfDF) and maintained in IDEC Pharmaceuticals'animal facility. Mice were housed in sterilized microbarrier units withsterilized bedding. Animal studies were performed in accordance with the“Guide for the Care and Use of Laboratory Animals” specified by theCommittee on Care of Laboratory Animal Resources Commission on LifeScience-National Research Council (Guide for the Care and Use ofLaboratory Animals, DHHS Publ. No. (NIH) 86-23, Bethesda, Md., NIH,1985).

Human PBMC: PBMCs were isolated from buffy coats obtained from a bloodbank by centrifugation through Ficoll-Hypaque (Histopaque-1077) asrecommended by the manufacturer (Sigma Diagnostics Catalog #1077-1).Lymphocyte preparation at the interface of the gradient were harvestedand washed three times in Hanks Balanced Salt Solution (HBSS)(Bio-Whittaker Catalog #10-527F). For each experiment PBMCs wereobtained from two separate donors and cultured separately in vitro.PBMCs were resuspended at 1-3×10⁶ cells/ml concentration in HB-Basalmedium plus 1% HB101 lyophilized supplement (Irvine Scientific Catalog#T000 & T151) containing 5% FCS plus 1000 IU/ml of IL-4 (Genzyme, Inc.Catalog #2181-01) and incubated for 48 hours at 37° C. with 5% CO₂.After incubation, the cells from different buffy coats were harvested,pooled and used to reconstitute SCID mice.

In Vivo Assay Conditions

Groups of mice (four to five per group) were injected withfifty-sixty×10⁶ lymphocytes in 200-300 μl volume of HBSSintraperitoneally (i.p.) on day zero. For the groups that receivedanti-CD23 antibody, on day zero, PBMCs were mixed with anti-CD23antibody (200 to 400 μg/mouse) before i.p., injection and the secondinjection was given on day seven. All mice received 5000 IU per mouse ofIL-4 i.p., between day zero to day five. A group which was not injectedwith antibody served as the control group. Mice were bled from aretro-orbital vein and the serum was analyzed for IgG and IgE on daysfourteen, twenty-one, twenty-eight and thirty-five by ELISA.

FIG. 8 shows that the primate anti-human CD23 monoclonal antibody 5E8 iseffective in inhibiting IL-4 induced IgE production in vivo in the SCIDmouse model.

Cloning and Expression of PRIMATIZED® Anti-Human CD23 MonoclonalAntibodies

In order to clone primate immunoglobulin variable domains, Poly A+ RNAwas separately isolated from approximately 2×10⁶ cells from the primateheterohybridomas secreting the anti-human CD23 monoclonal antibodies 6G5and 5E8 by using the Micro-FastTrack mRNA isolation Kit (InvitrogenCatalog #K1520-02) according to methods set forth by the manufacturer.

The first strand of cDNA was synthesized from the poly A+ RNA by usingthe cDNA Cycle Kit (Invitrogen Catalog #L1310-01) according toconventional methods.

The light and heavy chain variable regions of 6G5 and 5E8 were thenisolated by PCR from cDNA using PCR primers that were selected basedupon different consensus families of human immunoglobulins. 5′ primerswere selected which corresponded to the beginning of the leadersequences of the light and heavy variable region and 3′ primers wereselected which corresponded to the J region (The specific primers usedto PCR amplify the lambda light chain variable domain of 6G5, the kappalight chain variable domains of 5E8, and the heavy chain variabledomains of 6G5 and 5E8 are set forth in Tables 1-3). PCR was performedaccording to standard methods (30 cycles with 1 minute at 94° C., 1.5minutes at 54° C. and 2 minutes at 72° C. in a Hot start 100 tube (GibcoBRL Catalog #10332-013). PCR was set up in 50 μl reactions containing 5μl out of 80 μl cDNA (from 2×10⁶ cells) as a template, 2 μl of 5 nMDNTP, 1 μl of Taq polymerase, 5 μl of Taq polymerase buffer, 2 μl of the5′ primer (25 pmoles/μl), 2 μl of the 3′ primer (25 pmoles/μl), and 36μl of water. (Taq polymerase and buffer were obtained from StratageneCatalog #600131, DNTP from Boehringer

Mannheim Catalog #1581295.) A) Construction of the Plasmids N5LG1+6G5and N5LG4P+6G5 1) Cloning the Light Chain Variable Domain of PrimateMonoclonal Anti-Human CD23 Antibody 6G5 by PCR

The first PCR amplification of the light chain variable region from thecDNA of primate monoclonal antibody 6G5 showed bands which wereconsistent in situ with the lambda light chain variable region. Thesebands appeared in all reactions using the three different early leadersequence primers. (See Tables 1-3.) However, the PCR product obtainedusing primer 745 (Family 2) was considered more specific because of therelatively greater intensity of the PCR product band.

This PCR product was isolated using a Qiaquick Gel Extraction Kit(Qiagen Catalog #28704). The purified PCR fragment was digested with BglII and Avr II restriction endonucleases, and ligated into the mammalianexpression vector N5LG1 which was digested with the same restrictionendonucleases. Twenty microliters of the ligation mixture containing thepurified PCR product from one fifty microliter PCR reaction, 100 mgN5LG1 vector, two microliters of 10× ligation buffer (NEB Catalog #202S)and two microliters of T4 ligase (NEB Catalog # 202S), were thenincubated at 14° C. overnight.

The mammalian expression vector N5LG1 contains genetic sequences (e.g.,regulatory sequences, coding sequences) which provide for the expressionof four separate proteins in a mammalian cell. They are:

(i) a partial immunoglobulin light chain with the human lambda lightchain constant region and unique restriction endonuclease sites forinserting light chain variable domains;

(ii) a partial immunoglobulin heavy chain with the and human gamma 1chain constant region coding sequences and unique restrictionendonuclease sites for inserting heavy chain variable domains;

(iii) a neomycin phosphotransferase gene used to select for cells thathave incorporated the plasmid and are resistant to the antibioticGeneticin (Gibco BRL Catalog #10131-1209); and

(iv) a murine dihydrofolate reductase gene (DHFR) which provides for theselection and genomic amplification when cells are cultured in thepresence of methotrexate (MTX, Sigma Catalog #A-6770) (Reff et al.,Blood, 83:433-445 (1994).

After ligation, the mixture was digested using Pme I restrictionendonuclease, which digests the parent N5LG1 plasmid, but not the NSLG1plasmid which has been ligated to the light variable domain of 6G5.After digestion, the mixture was transformed into Epicurian coli®XL1-Blue competent cells (Stratagene Catalog #200249) as follows.

One hundred microliters of competent cells were mixed with 10 μl of theabove ligation mixture, set on ice for 30 minutes, then heated at 45° C.for 30 seconds. This mixture was placed on ice for 2 minutes, and 900 μlof SOC, prewarmed to room temperature, was then added. (SOC is LB brothGibco BRL Catalog #10855-013, plus 0.02 M MgCl₂, 0.02 M MgSO₄ and 0.02 MD-glucose.) After incubation at 37° C. for an hour, the mixture wascentrifuged at 4000 g for a minute, and 800 μl of supernatant discarded.The rest of the mixture was plated onto a LB agar (Gibco BRL Catalog#12945-044) dish containing 50 μg/ml ampicillin (Amp, Gibco BRL Catalog#13075-015). Plasmid DNA was isolated from individual colonies of E.coli that grew on the Amp plate by using the Wizard® Miniprep DNApurification system (Promega Catalog #A7510)

The isolated plasmid DNA was then characterized by digestion with Bgl IIand Avr II followed by agarose gel electrophoresis. An ethidium stainedDNA band of 400 bp was indicative of a potential successful cloning of alight chain variable domain.

To confirm this was an immunoglobulin light chain variable domain,sequencing was done using the Sequenase 7-Deaza-dGTP DNA Sequencing Kit(USB catalog #70990) with sequencing primers 607 and GE 108. (SeeSequencing Primers in Table 4.)

A second independent PCR amplification of the light chain from cDNA ofprimate monoclonal antibody 6G5 was effected using a 5′ primer earlyleader sequence of lambda light chain family 2 (primer 745) and the 3′ Jregion primer 926. (See Primers for PCR of the lambda light chainvariable domain of 6G5 in Tables 1-3.) The isolated PCR product (seetechnique above) was cloned into TA vector by using the Original TACloning (Kit (Invitrogen Catalog #K2000-01). The isolated miniprep DNA(see technique above) was examined under agarose gel electrophoresisafter digestion with EcoR I restriction endonuclease. The resultant PCRproduct comprised in the TA vector was then sequenced (as describedpreviously) using Sp6 and M13(−40) forward primers (See Sequencingprimers in Table 4). The resultant light chain sequence was identical tothat of light chain from the first PCR. This entire sequence of thelight chain variable domain of primate monoclonal anti-human CD23antibody 6G5 is presented below.

Light chain variable region of primate monoclonal antibody anti-humanCD23 6G5 Leader Met Ala Trp Thr Leu Leu Leu Val Thr Leu Leu Thr ATG GCCTGG ACT CTG CTC CTC GTC ACC CTC CTC ACT                          −1 GlnGly Thr Gly Ser Trp Ala CAG GGC ACA GGA TCC TGG GCT Mature Protein(Numbering is Kabat) Framework 1   1                              9  11Gln Ser Ala Pro Thr Gln Pro Pro Ser Val Ser Gly CAG TCT GCC CCG ACT CAGCCT CCC TCT GTG TCT GGG                          20          23 Ser ProGly Gln Ser Val Thr Ile Ser Cys TCT CCT GGA CAG TCG GTC ACC ATC TCC TGCCDR 1  24          27 27A 27B 27C  28 Thr Gly Thr Ser Asp Asp Val GlyGly Tyr Asn Tyr ACT GGA ACC AGC GAT GAC GTT GGT GGT TAT AAC TAT      34Val Ser GTC TCC Framework 2  35                  40 Trp Tyr Gln His HisPro Gly Lys Ala Pro Lys Leu TGG TAC CAA CAC CAC CCA GGC AAA GCC CCC AAACTC          49 Met Ile Tyr ATG ATT TAT CDR 2 50                      56Asp Val Ala Lys Arg Ala Ser GAT GTC GCT AAG CGG GCC TCA Framework 3 57          60 Gly Val Ser Asp Arg Phe Ser Gly Ser Lys Ser Gly GGG GTCTCT GAT CGC TTC TCT GGC TCC AAG TCT GGC    70                                       80 Asn Thr Ala Ser Leu ThrIle Ser Gly Leu Gln Ala AAC ACG GCC TCC CTG ACC ATC TCT GGG CTC CAG GCT                             88 Glu Asp Glu Ala Asp Tyr Tyr Cys GAG GACGAG GCT GAT TAT TAC TGT CDR 3  89  90                  95 95A  96  97Cys Ser Tyr Thr Thr Ser Ser Thr Leu Leu TGT TCA TAT ACA ACC AGT AGC ACTTTG TTA Framework 4  98     100                     106 106A 107 Phe GlyArg Gly Thr Arg Leu Thr Val Leu  Gly TTC GGA AGA GGG ACC CGG TTG ACC GTCCTA  GGT

2) Cloning the Heavy Chain Variable Domain of Primate MonoclonalAnti-Human CD23 Antibody 6G5 by PCR

The first PCR amplification of the heavy chain variable domain from cDNAof primate monoclonal antibody 6G5 was performed by using the set ofearly leader sequence primers described supra and the 3′ J region primerGE244. These primers are in Tables 1-3 infra. This reaction resulted ina 350 base PCR product. This 350 base product (purified as describedsupra), was digested with Nhe I and Sal I, and ligated into N5LG1 anddigested with the same endonucleases in the first PCR amplification. Theresultant ligation mixture was transformed into host cells using thesame techniques for cloning the light chain. Plasmid N5LG1 containingthe 350 base PCR product was then isolated and sequenced (usingsequencing primers 266 and 268). (These Sequencing primers are set forthin Table 4.)

Sequencing revealed that the PCR product contained only part of theheavy variable domain and comprised a deletion in its amino terminus(Sequence began at framework 2, codon 36).

A second independent PCR reaction was conducted to amplify and isolatethe heavy chain variable domain of primate monoclonal antibody 6G5 usinga 5′ early leader sequence primer for family 1 (MB1503) and a 3‘J’region primer GE244. (These primers are also contained in Tables 1-3.)The resultant PCR product was then cloned into the N5LG1 using the sametechniques described supra. Its sequence was found to be identical tothe first PCR product.

Therefore, in order to clone the whole heavy variable domain of 6G5including the missing 5′ terminus a new longer 3′ primer (MB1533) whichincluded the CDR3 and framework 4 regions of the 6G5 heavy variablechain was then used in a third independent PCR reaction with the family1 5′ primer (MB1503). (These primers are also contained in Tables 1-3.)

After PCR, a larger 420 base PCR product was observed on the agarosegel. This PCR product was isolated as described previously, and clonedinto a TA vector. The resultant PCR product contained in the TA vectorwas then sequenced. Sequencing revealed that this DNA contained thewhole heavy variable domain and that the 3′ part was identical to thatof previously cloned partial heavy chain variable domain from the firsttwo PCR reactions.

A fourth independent PCR was performed using the same primers as thethird PCR amplification. This resulted in a PCR product which wasisolated and cloned into the TA vector as described previously. Thesequence of the fourth independent PCR product was found to be identicalto that obtained in the third PCR amplification. This sequence, whichcomprises the heavy chain variable domain of primate monoclonalanti-human CD23 antibody 6G5, is presented below.

Heavy chain variable region of primate monoclonal antibody anti-humanCD23 6G5 Leader Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala ATG AAACAC CTG TGG TTC TTC CTC CTC CTG GTG GCA                         −1 AlaPro Arg Trp Val Leu Ser GCT CCC AGA TGG GTC CTG TCC Mature Protein(Numbering is Kabat) Framework 1   1                                 10Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Val Val CAG CTG CAG CTG CAG GAGTCG GGC CCA GGA GTG GTG                          20 Lys Pro Ser Glu ThrLeu Ser Leu Thr Cys Ala Val AAG CCT TCG GAG ACC CTG TCC CTC ACC TGC GCTGTC                     30 Ser Gly Gly Ser Val Ser TCT GGT GGC TCT GTCAGC CDR 1 31               35 35a Ser Ser Asn Trp Trp Thr AGT AGT AACTGG TGG ACC Framework 2 36               40 Trp Ile Arg Gln Pro Pro GlyLys Gly Leu Glu Trp TGG ATC CGC CAG CCC CCA GGG AAG GGA CTG GAG TGG     49 Ile Gly ATT GGA CDR 265               70                          60 Arg Ile Ser Gly Ser GlyGly Ala Thr Asn Tyr Asn CGT ATC TCT GGT AGT GGT GGG GCC ACC AAC TAC AAC            65 Pro Ser Leu Lys Ser CCG TCC CTC AAG AGT Framework 366               70 Arg Val Ile Ile Ser Gln Asp Thr Ser Lys Asn Gln CGAGTC ATC ATT TCA CAA GAC ACG TCC AAG AAC CAG         80      82 82a 82b 82c 83 Phe Ser Leu Asn Leu Asn Ser Val ThrAla Ala Asp TTC TCC CTG AAC CTG AAC TCT GTG ACC GCC GCG GAC            90               94 Thr Ala Val Tyr Tyr Cys Ala Arg ACG GCCGTG TAT TAC TGT GCC AGA CDR 3 95                 100 100a 100b 100c 100d 101 Asp Trp Ala Gln Ile AlaGly  Thr  Thr  Leu  Gly GAT TGG GCC CAA ATA GCT GGA  ACA  ACG  CTA  GGC102 Phe TTC Framework 4 103                         110         113 TrpGly Gln Gly Val Leu Val Thr Val Ser Ser TGG GGC CAG GGA GTC CTG GTC ACCGTC TCC TCA

3) Construction of Mammalian Expression Vectors

In order to insert the cloned heavy chain variable domains of 6G5 into amammalian expression vector, the heavy chain variable domain in the TAvector (obtained in the 3rd independent PCR) was digested with Nhe I andSal I and cloned into the N5LG1 vector which was digested with the samerestriction enzymes and which vector already contains the light chainvariable domain. The resultant mammalian expression vector was namedN5LG1+6G5.

To construct the N5LG4P+6G5 vector, both the light and heavy chainvariable domains were isolated from N5LG1+6G5 by digestion of Bgl II andAvr II, and Nhe I and Sal I respectively. The mammalian expressionvector N5LG4P vector is identical to the N5LG1 vector described above,except the human gamma 1 was replaced with a human gamma 4 constantregion containing a mutation of a serine to a proline in the hingeregion to increase stability of the immunoglobulin and improvepharmacokinetics in vivo (“P” mutation). The light chain variable domainwas cloned in the plasmid first and the heavy chain variable domain wascloned into the vector containing the light chain variable domain usingtechniques previously described. This mammalian expression vector wasnamed N5LG4P+6G5.

B. Construction of the Plasmids N5KG4P+5E8, N5KG1+5E8, N5KG4P+5E8N−, andN5KG1+5E8N− 1. Cloning the Light Chain Variable Domain of PrimateMonoclonal Anti-Human CD23 Antibody 5E8 by PCR

The first PCR reaction of the light chain variable domain from 5E8 cDNAwas carried out using a set of kappa early leader sequence primers andthe 3′ J region primer GE204. (See primers for PCR of the kappa lightchain variable domain of 5E8 in Tables 1-3). A 420 base PCR product wasobtained. The isolated 420 base PCR product was digested with Bgl II andBsiW I restriction endonucleases, cloned into the mammalian expressionvector N5KG4P and sequenced using GE108 and 377 primers (which arecontained in Table 4). The mammalian expression vector N5KG4P isidentical to the vector N5LG4P except it contains the human kappa lightchain constrant region is place of the human lambda light chain constantregion. Sequencing of this 420 polynucleotide DNA revealed that itcontains the entire kappa light chain variable domain.

A second independent PCR of the light chain variable region wasperformed using the 5′-family 1 primer GE201 and the 3′ primer GE204.(See primers for PCR of the kappa light chain variable domain of 5E8 inTables 1-3). The isolated PCR product was cloned into the TA vector(using methods previously described) and sequenced using Sp6 and T7promoter primers. Sequencing revealed that this PCR product wasidentical to that obtained from the first PCR. The entire sequence ofthe light chain variable domain of primate monoclonal anti-human CD23antibody 5E8 is presented below.

Light chain variable region of primate monoclonal antibody anti-humanCD23 5E8 Leader Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu ATG GACATG AGG GTC CCC GCT CAG CTC CTG GGG CTC                                     −1 Leu Leu Leu Trp Leu Pro Gly AlaArg Cys CTT CTG CTC TGG CTC CCA GGT GCC AGA TGT Mature Protein(Numbering is Kabat) Framework 1   1                                 10Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser GAG ATC CAG ATG ACC CAGTCT CCA TCT TCC CTG TCT                             20          23 AlaSer Val Gly Asp Arg Val Thr Ile Thr Cys GCA TCT GTA GGG GAC AGA GTC ACCATC ACT TGC CDR 1  24                      30             34 Arg Ala SerGln Asp Ile Arg Tyr Tyr Leu Asn AGG GCA AGT CAG GAC ATT AGG TAT TAT TTAAAT Framework 2  35                  45 Trp Tyr Gln Gln Lys Pro Gly LysAla Pro Lys Leu TGG TAT CAG CAG AAA CCA GGA AAA GCT CCT AAG CTC         49 Leu Ile Tyr CTG ATC TAT CDR 2 50                      56 ValAla Ser Ser Leu Gln Ser GTT GCA TCC AGT TTG CAA AGT Framework 3 57          60 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly GGG GTCCCA TCA AGG TTC AGC GGC AGT GGA TCT GGG    70                                       80 Thr Glu Phe Thr Leu ThrVal Ser Ser Leu Gln Pro ACA GAG TTC ACT CTC ACC GTC AGC AGC CTG CAG CCT                             88 Glu Asp Phe Ala Thr Tyr Tyr Cys GAA GATTTT GCG ACT TAT TAC TGT CDR 3  89  90                          97 LeuGln Val Tyr Ser Thr Pro Arg Thr CTA CAG GTT TAT AGT ACC CCT CGG ACGFramework 4  98     100                         107 Phe Gly Gln Gly ThrLys Val Glu Ile Lys TTC GGC CAA GGG ACC AAG GTG GAA ATC AAA

2) Cloning the Heavy Chain Variable Domain of Primate MonoclonalAnti-Human CD23 Antibody 5E8 by PCR

The first PCR of the heavy chain variable domain of 5E8 was performedusing a set of 5′ early leader heavy chain sequence primers and the 3′primer GE210. (See primers for PCR of the heavy chain variable domain of6G5 and 5E8 in Table 1). A 420 base PCR product appeared in the family 3primer reaction. The PCR product was purified and then digested with NheI and Sal I and cloned into the mammalian expression vector N5KG4Pvector (as described previously). The PCR product was sequenced usingthe 268 and 928 primers. (See sequencing primers in Table 4.)

A second independent PCR of the heavy chain variable domain of 5E8 wasperformed using the family 3 5′ primer GE207 and the 3′ primer GE210.(See primers for PCR of the heavy chain variable domain of 6G5 and 5E8in Tables 1-3). The isolated PCR product was cloned into a TA vectorusing the same techniques previously described and sequenced by usingSp6 and T7 primers. Sequencing revealed that the TAC at codon 91 hadbeen changed into TGC.

In order to determine the appropriate codon at 91, a third independentPCR was performed using the same primers as the second PCR (see above).The PCR product was again cloned into a TA vector and sequenced usingSp6 and T7 primers. The sequence was found to be identical to the heavychain variable sequence obtained in the first PCR. Therefore, the TGC atposition 91 in the second independent PCR product is apparently theresult of an error introduced during PCR. This entire sequence of theheavy chain variable domain of primate monoclonal anti-human CD23antibody 5E8 is presented below.

Heavy chain variable region of primate monoclonal antibody anti-humanCD23 5E8 Leader Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Pro ATG GAGTTT GGG CTG AGC TGG GTT TTC CTT GTT CCT                         −1 LeuLeu Lys Gly Val Gln Cys CTT TTG AAA GGT GTC CAG TGT Mature Protein(Numbering is Kabat) Framework 1   1                                 10Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ala GAG GTG CAG CTG GTG GAGTCT GGG GGC GGC TTG GCA                              20 Lys Pro Gly GlySer Leu Arg Leu Ser Cys Ala Ala AAG CCT GGG GGG TCC CTG AGA CTC TCC TGCGCA GCC                      30 Ser Gly Phe Arg Phe Thr TCC GGG TTC AGGTTC ACC CDR 1 31               35 35a 35b Phe Asn Asn Tyr Tyr Met AspTTC AAT AAC TAC TAC ATG GAC Framework 2 36               40 Trp Val ArgGln Ala Pro Gly Gln Gly Leu Glu Trp TGG GTC CGC CAG GCT CCA GGG CAG GGGCTG GAG TGG      49 Val Ser GTC TCA CDR 250       52 52A  53                          60 Arg Ile Ser Ser Ser GlyAsp Pro Thr Trp Tyr Ala CGT ATT AGT AGT AGT GGT GAT CCC ACA TGG TAC GCA            65 Asp Ser Val Lys Gly GAC TCC GTG AAG GGC Framework 36                70 Arg Phe Thr Ile Ser Arg Glu Asn Ala Asn Asn Thr AGATTC ACC ATC TCC AGA GAG AAC GCC AAC AAC ACA 80              82 82a 82b 82c 83 Leu Phe Leu Gln Met Asn Ser Leu ArgAla Glu Asp CTG TTT CTT CAA ATG AAC AGC CTG AGA GCT GAG GAC            90          94 Thr Ala Val Tyr Tyr Cys Ala Ser ACG GCT GTCTAT TAC TGT GCG AGC CDR 3  95                 100 101 Leu Thr Thr GlySer Asp Ser TTG ACT ACA GGG TCT GAC TCC Framework 4103                         110         113 Trp Gly Gln Gly Val Leu ValThr Val Ser Ser TGG GGC CAG GGA GTC CTG GTC ACC GTC TCC TCA

3) Construction of Mammalian Expression Vectors

The heavy variable domain in N5KG4P was digested with Nhe I and Sal I,purified, and cloned into NSKG4P which contains the light chain variabledomain of 5E8. This plasmid was then digested with the restrictionendonucleases as previously described. This resulted in a vectorcontaining both the light and heavy variable domain of 5E8. This vectorwas named N5KG4P+5E8. The heavy and light variable domains of N5KG4P+5E8were then both inserted into the mammalian expression vector N5KG1 tocreate the N5KG1+5E8 vector.

4) Alteration of an Amino Acid in the Heavy Chain Variable Region ofPrimate Monoclonal Antibody 5E8 by Site Specific Mutagenesis andConstruction of Mammalian Expression Vectors

Based upon the sequence of 5E8 heavy variable domain, there is apotential glycosylation site of the immunoglobulin at asparagine codon75. This potential glycosylation site corresponds to a conservedasparagine-linked glycosylation motif having the following tripeptidesequence: (Asp)—(Any amino acid except proline)—(Serine or threonine).Therefore, a glycosylation mutant of 5E8, which would be unable to beglycosylated at this position because of modification of thisglycosylation motif, was generated by replacing the asparagine codon 75with a lysine (which is found in many human immunoglobulins at thisposition). Site specific mutagenesis was effected by the followingmethods.

A first PCR was done using N5KG4P+5E8 as a template and a 3′ primer(corresponding to codon 71 to 79) and which contains a mutation at codon75 (AAC changed to AAG, Primer MB1654, and a 5′ primer at the beginningof the leader sequence (Primer MB1650). (See PCR Primers Used for theGeneration of a Glycosylation Mutant of the Heavy Chain Variable Region5E8 set forth in Table 5).

A second PCR was performed on the same template by using a 5′ primer(corresponding to codon 71 to 79) containing the same mutation (PrimerMB1653) and a 3′ primer from the end of framework 4 (Primer MB1651) (SeePCR Primers Used for the Generation of a Glycosylation Mutant of theHeavy Chain Variable Region of 5E8 in Table 5.)

These two PCR products were isolated and mixed in equal molar ratios. Athird independent PCR was then carried out by using the mixture of thefirst and second PCR products as a template with a 5′ primer used in thefirst PCR (MB1650) and a 3′ primer used in the second PCR (MB1651). (SeePCR Primers Used for the Generation of a Glycosylation Mutant of theHeavy Chain Variable Region in Table 5.) The PCR product obtained inthird PCR was found to contain the heavy variable domain coding regionof 5E8 wherein the asparagine 75 had been changed to lysine.

The third PCR product was purified, digested with restrictionendonucleases Sal I and Nhe I, and cloned into the N5KG4P containing thelight variable domain only. The PCR product was sequenced to confirmthat it comprised the mutant heavy variable domain. This mammalianexpression plasmid was named NSKG4P+5E8N−.

To construct of N5KG1+5E8N−, both light and heavy variable domains fromN5KG4P−5E8N− were digested with Bgl II and BsiW I, and Nhe I and Sal Irespectively. The light variable domain was cloned first and then theheavy variable domain was inserted into the mammalian expression plasmidN5KG1, to create the plasmid N5KG1+5E8N−.

TABLE 1 Primers for PCR of the kappa light chain variable domain of 5E8NAME FAMILY Light chain Vk-early leader 5′ (Bgl II)                          −22 −21 −20 −19 −18     −17 −16     −15 −14GE201 5′ AT CAC AGA TCT CTC ACC ATG GAC ATG AGG GTC     CCCGCT     CAG  3′ 1 GE200 5′ AT CAC AGA TCT CTC ACC         ATG AGGCTC     CCT GCT     CAG  3′ 2 GE202 5′ AT CAC AGA TCT CTCACC         ATG GAA (A/G)CC CCA GC(T/G) CAG  3′ 3 GE203 5′ AT CACAGA TCT CTC ACC         ATG GTG TTG     CAG ACC     CAG GTC 3′ 4 Lightchain Vk-3′ primer (BsiW I)           113 112 111 110 109 108 107106   105   104 103 GE204 5′ GG TGC AGC CAC CGT AGC TTT GAT (C/T)TCCA(G/C) CTT 3′

TABLE 2 Primers for PCR of the lambda light chain variable domain of 6G5NAME FAMILY Light chain V1-early leader 5′ (Bgl II)                           −20  −19    −18     −17     −16    −15 7445′ AT CAC AGA TCT CTC ACC ATG (G/A)CC TG(G/C) TCC     CCT     CT 3′ 1745 5′ AT CAC AGA TCT CTC ACC ATG GCC     TGG     (A/G)CT C(T/C)G CT 3′2 910 5′ AT CAC AGA TCT CTC ACC ATG GC(A/C) TGG     A(T/C)C CCT     CTC3′ 3 Light chain V1-3′ primer (Avr II)               110 109 108 107 106105 104 926 5′ (AC)10 CTT GGG CTG ACC TAG GAC GGT 3′

TABLE 3 Primers for PCR of the heavy chain variable domains from 6G5 and5E8 NAME Family Heavy chain-early leaders 5′ (Sal I)                               −20 −19 −18 −17 −16 −15 MB1503 5′ GCG ACTAAG TCG ACC ATG GAC TGG ACC TGG     3′ 1 MB1502 5′ GCG ACT AAG TCG ACCATG AAA CAC CTG TGG     3′ 2, 4 GE207 5′ GCG ACT AAG TCG ACC ATG GAG TTTGGG CTG AGC 3′ 3 GE208 5′ GCG ACT AAG TCG ACC ATG GGG TCA ACC GCC ATC 3′5 GE209 5′ GCG ACT AAG TCG ACC ATG TCT GTC TCC TTC CTC 3′ 6 Heavychain-3′ primer (Nhe I)                           120 119 118 117 116 115 114 113 112 110 110GE244 5′ GC CAG GGG GAA GAC CGA TGG GCC CTT GGT GCT AGC TGA GGA GAC GG3′ GE210 5′                     GA TGG GCC CTT GGT GCT AGC TGA GGA GACGG 3′ MB1533 5′                                    GGT GCT AGC TGA GGAGAC GGT                         109 108 107 106 105 104 103 101 100  99                        GAC CAG GAC TCC CTG GCC CCA GAA GCC TAG 3′

TABLE 4 Sequencing Primers Sp6 primer 5′ AT TTA GGT GAC ACT ATA 3′ M13(−40) Forward 5′ GTT TTC CCA GTC ACG A 3′ Primer T7 Promoter Primer5′ AT ATA CGA CTC ACT ATA GGG 3′ GE 108 Primer 5′ CCG TCA GAT CGC CTGGAG ACG CCA 3′ 377 Primer 5′ GCA GTT CCA GAT TTC AAC TG 3′ 607 PRIMER5′ CCA GGC CAC TGT CAC GGC TTC 3′ 266 PRIMER 5′ CAG AGC TGG GTA CGT CCTCA 3′ 268 PRIMER 5′ GCC CCC AGA GGT GCT CTT GG 3′ 876 PRIMER 5′ ACA CAGACC CGT CGA CAT GG 3′ 928 PRIMER 5′ GCT CTC GGA GGT GCT CCT GG 3′

TABLE 5 PCR Primers Used for the Generation of a Glycosylation Mutant ofthe Heavy Chain Variable Region of 5E8                SalI   −20 −19 −18 −17 −16 MB 1650 5′ ACA GAC CCG TCG ACC ATG GAG TTT GGGCTG 3′                Nhe I     118 117 116 115 114 113 112 111 110 MB1651 5′ CCC CTT GGT GCT AGC TGA GGA GAC GGT 3′        71  72  73  74  75  76  77  78  79 MB 1653 5′ AGA GAG AAC GCCAAG AAC ACA CTG TTT   3′         79  78  77  76  75  74  73  72  71 MB1654 5′ AAA CAG TGT GTT CTT GGC GTT CTC TCT   3

C) Construction of Gamma-3 Vectors

Numerous methods exist for conversion of murine antibodies to chimerasin which the heavy and light chain constant regions are substituted withhuman versions or in which all but the CDRs (complementary determiningregions) are substituted with their human equivalents. (See Biochem. J.281:317, 1992; Proc. Nat. Acad. Sci. USA 86:10029, 1989; MethodsEnzymol. 178:515, 1989; Cancer Res. 51:181, 1991; Biotechniques 7:360,1989; J. Immunol. 143:3589, 1989; Int. J. Cancer 44:424, 1989; Proc.Nat. Acad. Sci. USA 86:3833, 1989).

Further, the previous sections specifically demonstrate thatconstruction of vectors for expression of PRIMATIZED® gamma-1 andgamma-4 antibodies may be accomplished by amplifying the DNA encodingthe light and heavy chain variable regions using PCR, and cloning theseregions into an expression vector or vectors encoding the appropriatehuman constant region domains. Thus, it should be clear that similarconstructs may be made which encode other human constant region domains,so long as appropriate mammalian expression vectors are available whichalso contain cloning sites which allow insertion of the amplifiedvariable region DNA from the primate antibodies.

It should also be apparent that the PCR primers used to amplify thevariable region DNA may be designed to accommodate various restrictioncloning sites depending on the vector used.

Vectors encoding the constant region domain from human IgG3 areavailable in the art, and may be used for constructing the PRIMATIZED®antibodies of the present invention. For instance, Co et al. have usedseparate vectors expressing human light and heavy chain constant regionsfor constructing chimeric and humanized antibodies, and provide heavychain vectors for both human gamma-1 and gamma-3. For cloning thevariable domain regions of the antibody of interest into these vectors,an XbaI restriction site is conveniently located just before theconstant regions in the Jk⁴ and the JH³ intronic segments for the lightand heavy chains, respectively (Co et al., 1996, Cancer Res. 56:1118-1125; Co et al., 1992, J. Immunol. 148: 1149-1154).

Thus, by designing PCR primers which incorporate XbaI restriction sites,or sites which create the same single stranded overhangs as the XbaIrestriction enzyme (i.e. NheI, SpeI), the variable regions from aprimate antibody may be associated with the human gamma-3 constantregion domain using the vectors described in Co et al. Alternatively,linkers or adaptors may be used to facilitate cloning.

It is also possible to construct a cDNA version of the human gamma-3constant region using DNA synthesis techniques and insert it into theN5KG1 vectors described herein in place of the human gamma-1 constantregion between the NheI and BamHI sites. Depending on which restrictionsites are most convenient for cloning the particular variable region ofinterest, there are also other gamma-3 vectors which have been describedin the art, and any of these may be used to construct and express thegamma-3 antibodies of the present invention. (See, for example, Parrenet al., 1992, J. Immunol. 148(3): 695-701; Steplewski et al., 1988,Proc. Natl. Acad. Sci. USA 85: 4852-4856; and U.S. Pat. No. 4,975,369,herein incorporated by referenced).

D) Variations of PRIMATIZED® Antibodies

The present invention also encompasses PRIMATIZED® antibodies containingmutations, substitutions or deletions of the constant region. Suchmutations may be constructed using site-directed mutagenesis techniquesas described above for the glycosylation mutant, which are well-knowntechniques for those of skill in the art.

The mutated antibodies of the present invention are designed with thepurpose of creating a desired change in the level of therapeuticefficiency, i.e. in FcR binding. However, it should be understood thatany mutation in the constant region of the antibodies should not alterthe basic effector functions as mediated by the gamma-1 or gamma-3constant region domains. It may also be possible to alter othersubclasses of gamma antibodies by mutagenesis such that they havesimilar effector functions as an antibody containing a gamma-1 or agamma-3 domain. Such antibodies are also encompassed by the presentinvention.

For example, the regions of the IgG constant region involved in FcRbinding and interaction with the C1q complement component have beencharacterized, and mutations have been identified which either increaseor decrease binding affinity. As disclosed in U.S. Pat. No. 5,648,260,herein incorporated by reference, changing Leu 235 to Glu in the humanIgG3 constant region destroys the interaction of the mutant for thehuman Fc gamma R1 receptor. Furthermore, mutations on adjacent or closesites in the hinge link region (i.e. replacing residues 234, 236 or 237by Ala) indicate that alterations in residues 234, 235, 236 and 237 atleast affect affinity for the Fc gamma R1 receptor.

Similarly, in U.S. Pat. No. 5,348,876, also incorporated by reference,it was shown that by decreasing the length of the hinge region of thehuman IgG3 constant region and altering the amino acid sequence of thisregion, Clq binding and thus complement mediated cytolysis were furtherimproved. In fact, the variants bound more Clq than wild type IgG3 andmuch more than IgG1.

Thus, it is clear from the art that mutations may be used to createtherapeutic antibodies with decreased affinities for human Fc receptors,perhaps for the treatment of more mild medical conditions. In addition,mutations may be identified which increase effector function activity,thereby leading to stronger versions for therapeutic use. It should berecognized that manipulations of gene sequence to create alterations indrug efficiency are envisioned, and well within the spirit and scope ofthe present invention.

E) Expression of PRIMATIZED® Antibodies in Chinese Hamster Ovary Cells

In order to isolate the PRIMATIZED® antibodies, the vectors encoding theantibodies were expressed in Chinese Hamster Ovary cells using thefollowing protocol.

A large scale plasmid DNA was purified using the WIZARD® Maxipreps DNAPurification System (Promega Catalog #A7421). The purified DNA wasdigested with Ssp I and BspLU11 I, precipitated with ethanol once, andresuspended in sterile TE.

Purified, endonuclease restricted plasmid DNA was then introduced intoChinese hamster ovary (CHO) dihydrofolate reductase minus DG44 cellsusing electroporation. The electroporation technique used is describedbelow.

Approximately 1.6×10⁸ CHO cells were spun in an appropriate size sterileCorning tube for one minute at 1000 RPM. The media was removed and thecells were washed in fifteen milliliters of sterile ice cold SBS(sterile sucrose buffered solution is 272 mM sucrose, 7 mM sodiumphosphate pH 7.4, 1 mM MgCl₂) and spun for 5 minutes at 1000 RPM. TheSBS was removed and cells were suspended using fresh ice cold sterileSBS at a cell concentration of 1×10⁷ cells were per ml and left on icefor 15 minutes. The BTX 600 electroporator was turned on and preset at230 volts, with the maximum voltage knobs being set at 500volts/capacitance & resistance. The capacitance was set at 400microfaradays and the resistance was set at 13 ohms (setting R1).

Plasmid DNA (4 μg DNA or 2 μg DNA) and 0.4 ml of cells (4×10⁶ cells)were then placed in BTX 0.4 ml cuvettes (BTX Catalog #620). The cellswere shocked by placing the cuvette into the BTX 600 stand and pressingthe automatic charge & pulse button. Approximately 20 separateelectroporations were performed with each mammalian expression plasmid.

After shocking, the cuvettes were left at ambient temperature forfifteen minutes. The cells and DNA were from each cuvette wereresuspended in 20 ml of CHO-SSFMII containing no hypoxanthine orthymidine (Gibco BRL Catalog #31033-012) to which HT supplement (100×supplement is 10 mM sodium hypoxanthine, 1.6 mM thymidine Gibco BRLCatalog #11067-014) had been added. The cells from a singleelectroporated cuvette were then plated into a 96 well plates (200Ml/well) and placed into a 37° C. CO₂ incubator. Selection was startedtwo or three days later by changing the media to the above media withthe addition of 400 mg/ml of Geneticin® (G418, Gibco BRL Catalog#10131-019). The cells were grown at 37° C. and the cell media werechanged every 3-5 days. After sixteen days G418 resistant clonesappeared in the wells and the supernatant was assayed for antibodyexpression by ELISA. The highest expressing clones were then expandedindividually. Monoclonal antibodies were purified as described below.

Immunoglobulin ELISA

Plates (Immulon 2, Dynatech Laboratories, Inc. Catalog #011-010-3455)are coated overnight at 4° C. with 200 ng unlabeled goat anti-human IgGantibody at 100 μl/well. This is effected using twenty milliliters ofunlabeled goat anti-human IgG/10 mls Coating Buffer/plate (BoehringerMannheim Ab Catalog #605 400). (1:500 dilution of ˜1 mg/ml stock.) Thecoating buffer is then removed from the plates and dried using a papertowel. One hundred microliters of a dilution buffer/well is then added.

Antibody solutions and standards (100 ng/ml-2.5 ng/ml) are then added induplicate at 100 μl/well directly to the 100 ml dilution buffer. Theantibody solutions and standards are contained in dilution buffer. Theresultant solutions are then incubated for at least 1 hour at 37° C.

After incubation, the contents of each plate are removed and the platesare washed with tap water five times. The plates are then dried on apaper towel.

After drying of the plates, a second antibody is then added at 100μl/well. This second antibody is either goat anti-human Kappa-HRPO:added at 1/10,000 dilution or 1 μl Ab/10 mls dilution buffer/plate,available from Southern Biotechnology Associates, Inc. Catalog #2060-05or a goat anti-human Lambda-HRPO; used at 1/20,000 dilution or 1 μlAb/20 mls dilution buffer/2 plates (available from SouthernBiotechnology Associates, Inc. Catalog #2070-05).

The antibody and contents of the plate are allowed to incubate for onehour at 37° C. After incubation the contents of each plate are removed.The plates are again washed five time with tap water, and the washedplates are dried. To the dried plates is then added HRPO substrate (TMBMicrowell—two component) in an amount of 100 μl/well. (Five millilitersof TMB Peroxidase Substrate+five milliliters of Peroxidase SolutionB/plate (Kirdgaard and Perry Labs, TMB Microwell two component reagentsCatalog #50-76-00).

The reaction is stopped by the addition of one hundred microliters of 2MH₂SO₄ to each well when the weakest standard (2.5 ng/ml) is visible overbackground. The optical density of wells in plates is then read using aplate reader, e.g., Molecular Devices Emax precision microplate readerset at wavelength: OD 450 and OPT2 (OD 540).

ELISA Buffers

Coating Buffer Sodium Carbonate  0.8 gram/liter Sodium Bicarbonate 1.55gram/liter Adjust pH to 9.5 with ^(~)1 ml 1N HCl Dilution Buffer 0.5%Nonfat Dry Milk in PBS plus 0.01% Thimerosal (5 gm/L) (100 mg/L)

Examples of ELISA values obtained using the above-described assay areset forth below.

Standard OD 450 OD450 Average 100 ng/ml 0.805 0.876 0.841 50 ng/ml 0.3950.472 0.434 25 ng/ml 0.213 0.252 0.233 10 ng/ml 0.089 0.105 0.097 5ng/ml 0.054 0.055 0.055 2.5 ng/ml 0.031 0.035 0.033 0 ng/ml 0.004 0.0060.005

Standards in Dilution Buffer

Appropriate dilution of stock AB (sterile filtered in normal saline,protein determination by OD) to give 1 mg/ml

Example

Chimeric monkey/human anti-CD4 (CE9.1) is 4.18 mg/ml

24 μl of above into 76 μl Dilution Buffer is 1 mg/ml

50 AI Stock Ab (1 mg/ml) into 450 μl Dilution Buffer (D.B.) is 100 μg/ml

50 μl of above mixture into 450 μl D.B. is 10

200 μl of above mixture into 1.8 mls D.B. is 1 μg/ml

1 ml of above mixture into 9 mls D.B. is 100 ng/ml *

5 ml of above mixture into 5 ml D.B. is 50 ng/ml *

5 ml of above mixture into 5 ml D.B. is 25 ng/ml *

4 ml of above mixture into 6 ml D.B. is 10 ng/ml *

5 ml of above mixture into 5 ml D.B. is 5 ng/ml *

5 ml of above mixture into 5 ml D.B. is 2.5 ng/ml *

* Standards used in the ELISA

Antibody Purification by Protein A Procedure

The culture supernatant is centrifuged to remove cells and debris. Thecentrifuge is then filtered through a 0.2 μm filter. A protein Asepharose Fast flow column (recombinant protein A Sepharose Fast floe)(Pharmacia Biotech Catalog #71-5000-09) is then prepared andequilibrated using PBS (pH 7.4).

The supernatant is loaded on the column at an appropriate flow rate(e.g., 2 ml/min). After loading, the column is washed with 10 columnvolume of PBS (pH 7.4). The antibody is eluted from the column using anelution buffer (0.2M acetic acid, 0.1 M glycine pH 3.5) at 1 ml/min flowrate. One milliliter fractions/tube including 100 μl of Tris are thencollected. A spectrophotometer absorbance reading is then taken at 280nm. The antibody fractions are then collected and dialyzed against PBS(pH 7.9) overnight. The dialysate is then sterilized by filtrationthrough a 0.22 μm membrane and stored at −20° C.

Assay Results

The PRIMATIZED® human gamma-4 anti-human CD23 antibodies which aredescribed supra, were purified and assayed for induced IgE inhibitoryactivity in vitro. These results are contained in FIGS. 3 and 5. Thiswas effected using the in vitro IL-4 IgE assay described supra.

These assay results surprisingly indicated that both human gamma-4anti-human CD23 antibodies were not as active as the correspondingprimate anti-human CD23 antibodies, i.e., they did not significantlyinhibit induced IgE production in vitro.

However, because primate 5E8 and p5E8G4P have a potential asparaginelinked glycosylation site in the heavy chain variable region, theeffects of glycosylation at this site were investigated. (It was foundthat both these antibodies contain N-linked oligosaccharides at thissite. (Data not shown.)) Therefore, in order to prevent glycosylation,the asparagine in the glycosylation site was changed to a lysine inorder to eliminate carbohydrate addition. This mutated antibody wasnamed p5E8G4PN−. Assay results demonstrated that this antibody behavedidentically to p5E8G4P in the IL-4 IgE assay (see FIG. 3) and alsoexhibited an identical apparent affinity Kd for human CD23. (See FIG.4.) Therefore, these results indicated that the difference in IgEinhibition observed from the 5E8 gamma 4 PRIMATIZED® antibody incomparison to primate 5E8 antibody was not attributable to glycosylationdifferences.

The three primate antibodies (p5E8G4P, p5E8G4PN−, and pGG5G4P) were thenexpressed as human gamma-1 versions using substantially the samemethodology. All three human gamma-1 anti-human CD23 antibodies,respectively designated p5E8G1, p5E8GlN− and pGG5G1, were found to beactive in the in vitro IL-4/IgE assay (FIGS. 3 and 5).

p5E8G1 was found to be statistically more suppressive than p5E8G4P at aconcentration of 0.3 Mg/ml (P[T,t] one tail+0.0055) and at 3 μg/ml(p[T<t] one tail+0.0019). In addition, p5E8G1N− is statistically moresuppressive than p5E8G4PN− at both 0.3 μg/ml (p[T<t] one tail+0.0392)and at 3 μg/ml (p[T<t] one tail+0.0310) (FIG. 3).

Similarly, p6G5G1 completely inhibited induced IgE production at 3mg/ml, while p6G5G4P did not. (These results are in FIG. 5).

Thus, these results suggested that an active Fc region, in particularthat of human gamma-1, is significant for induced IgE inhibition byanti-human CD23 antibodies. These results also suggest that humananti-CD23 antibodies containing human gamma-3 constant regions will besignificant for induced IgE inhibition because a gamma-3 constant regionbinds to the same FcγR receptors that gamma-1 binds to, and will likelymediate the same result.

EXAMPLE 2

To confirm our hypothesis as to the involvement of the Fc effectorportion in IgE inhibition of anti-human CD23 antibodies, a third primateantibody, designated 2C8, also shown to inhibit IgE in in vitro) wasconverted to a F(ab′)₂. IgE inhibitory activity was determined using thesame IL-4/IgE assay described previously.

Materials

The following materials were used in this example.

ImmunoPure F(ab′)₂ Preparation Kit (Pierce Catalog #

44888)

digestion buffer: 20 mM sodium acetate buffer, pH 4.5

0.1 M citric acid, pH 3.0 (adjust pH with NaOH)

0.1% sodium azide in water

dialysis tubing; 50,000 MW cutoff (Spectra Por Catalog #132 128)

shaking water bath capable of maintaining 37° C.

polystyrene culture tubes, 17×100 mm (Fisher Catalog #14-956-6B)

BCA protein assay (Pierce Catalog #23224)

Centricon-50 concentrators (Amicon Catalog #4225).

Equilibration of Immunobilized Pepsin

0.25 milliliters of the 50% slurry of Immobilized Pepsin is added to a17×100 mm test tube (0.125 ml of gel). Four milliliters of digestionbuffer are then added. The pepsin is then separated from the bufferusing the serum separator. The buffer is then discarded and the washprocedure repeated using another four milliliters of buffer. Theimmobilized pepsin is then resuspended in 0.5 ml of digestion buffer.

Preparation of Immobilized Protein A Column

Protein A AffinityPak® columns and ImmunoPure Binding and Elutionbuffers are brought to room temperature.

Preparation of 2C8 F(ab′)₂ Fragments

2C8 F(ab′)₂ fragments are prepared by methods well known in the antibodyart. The inventors elected to use a commercially available kit,ImmunoPure F(ab′)₂ Preparation Kit (Pierce Catalog #44888), Using theManufacturer's Protocols.

Ten milligrams of lyophilized 2C8 antibody were dissolved in onemilliliter of a digestion buffer (20 mM sodium acetate buffer, pH 4.5).One milliliter of the antibody containing sample was than added to atube containing immobilized pepsin.

The antibody and immobilized pepsin were then incubated for four hoursin a high speed shaker water bath at 37° C. (at high speed), taking careto maintain the mixing constant during the incubation.

The resultant solubilized F(ab′)₂ and Fc fragments and the undigestedIgGs were then recovered from the immobilized pepsin gel using a serumseparator. The crude digest is then decanted into a clean tube.

In order to enhance recovery of F(ab′)₂ fragments, the immobilizedpepsin desirably is then washed with 1.5 of milliliters of theImmunoPure IgG binding buffer. The wash is then added to the crudedigest.

The antibody fragments were then recovered using a protein A column.This is effected by opening an immobilized protein A column. Care istaken to avoid air bubbles from being drawn into the gel. The storagesolution (which contains 0.02% sodium azide) is discarded.

The immobilized protein A column was then equilibrated using twelvemilliliters of binding buffer (contained in ImmunoPure Preparation Kit).The column was then transferred to a 17×100 nm test tube contained inthe kit (labeled “F(ab′)₂”) to collect eluate.

Three milliliters of the crude digest was then applied to a column andare allowed to flow completely into the gel. The use of AffinityPak™columns is desirable as these columns stop flowing automatically whenthe level reaches the top frit.

The column is then washed using six milliliters of binding buffer. Theeluate which contains F(ab′)₂ fragments was then collected. This eluatealso contains small Fc fragments that can no longer bind protein A(which are not bound to the Protein A column). However, the substantialportion thereof was eliminated by dialysis.

Dialysis was effected by taking the F(ab′)₂ containing eluate anddialyzing the eluate against pH 7.4 phosphate buffered saline, usingdialysis tubing with a molecular weight cut-off of 50,000 so as toeliminate the small Fc fragment containments (Spectra Pur. Catalog #132128).

This resulted in a F(ab′)₂ fraction having an optical density of 280 nmof 0.707 (6 ml). After dialysis and concentration with Centricon-50concentrators (Amicon Catalog #4225), the 2C8 F(ab′)₂ product wasassayed for protein content using a BCA protein assay (Pierce Catalog#23224). The protein content was found to be 3.76 mg per milliliter.

The 2C8 F(ab)₂'s were assayed for IgE inhibitory activity and were foundto be substantially incapable of inhibiting IgE production in the samein vitro assays described previously. These results are contained inFIG. 6. In fact, the F(ab′)₂ was found to antagonize the suppressiveeffects of induced IgE on the monoclonal antibody 2C8. These results arein FIG. 7.

EXAMPLE 3

The PRIMATIZED® gamma 1 and gamma 4P versions of primate monoclonal 6G5were both evaluated for their effect on inhibition of induced IgEproduction in vivo in the SCID mouse model described previously.p5E8G1N− was found to be as efficient as primate 5E8 in inhibitinginduced IgE. (See FIGS. 8 and 9). While neither primate 6G5 nor theprimatized p6G5G4P were effective at inhibiting induced IgE in vivo,primatized p6G5G1 inhibited induced IgE production. (See FIGS. 9 and10.) These results further substantiate our conclusion that an active Fcregion is significant to the ability of an anti-human CD23 antibody toeffectively inhibit induced IgE production.

Proposed Mechanism

Although the results of the present invention conclusively demonstratethat an active Fc region is significant to the ability of an anti-humanCD23 antibody to effectively inhibit induced IgE production, themolecular mechanism has not been elucidated. However, several hypothesesmay be made.

Firstly, it may be that the anti-CD23 antibodies of the presentinvention function to trigger a similar signal transduction pathway asis apparently triggered by cross-linking B cell receptor with anti-BCRgamma-1 antibodies. It has been hypothesized that the down-regulation ofantigen receptor signaling in this case results from coligation of Fcgamma RII and surface Ig (B cell receptor) on the same B cell, leadingto down regulation of Ig expression. (D'Ambrosia et al., 1995, Science,268:293-297; Ono et al., 1997, Cell 90: 293-301). Indeed, CD23 isupregulated on the surface of IgE-expressing B cells, so it is possiblethat coligation of CD23 and the FcγRII-B receptor via the gamma-1constant region results in a similar signal transduction pathway.

Alternatively, inhibition of induced IgE expression may be occurringthrough cross-linking of surface IgE on B cells at the same time as theFc region interacts with an appropriate FcγR receptor on another celltype, i.e. a killer T cell, which may then “instruct” the B cell toundergo apoptosis, or lead to cell death in some other manner (i.e.phagocytosis). For instance, U.S. Pat. No. 5,543,144, hereinincorporated by reference, proposes a similar mechanism for inhibitingIgE-expressing B cells with anti-IgE antibodies. As discussed therein,antibodies of certain IgG subclasses, such as mouse IgG2a and human IgG1and IgG3, can mediate ADCC (Antibody Dependent Cellular Cytotoxicity)carried out by certain Fc receptor-bearing phagocytic leukocytes. Forexample, OKT3, a mouse IgG2a monoclonal antibody specific for human Tcell surface antigen (which was the first monoclonal antibody productapproved by the FDA for marketing as a therapeutic agent) is used inpatients to provide rapid depletion of T cells in the blood and toinduce an immunosuppressed state (for kidney transplantation). (Russell,P. S. et. al, Transpl. Proc. 17:39-41 (1985)). OKT3, at a dosage of 5mg/day/subject, can completely deplete circulating T cells. Themonoclonal antibodies described in U.S. Pat. No. 5,543,144, especiallyin the form of mouse gamma 2a antibodies or human or humanizedantibodies bearing human gamma-1 or gamma-3 chains, are proposed todeplete IgE-expressing B cells by the ADCC mechanism in a similarmanner.

But whatever the actual molecular mechanism for inhibiting IgEexpression using anti-CD23 antibodies may be, it is clear that the Fcregion, and accordingly effector function, of anti-CD23 antibodies areimportant considerations. As discussed previously, since complement isnot present in the in vitro assays of the present invention, thephenomenon likely involves one or more FcγR receptors. Once the relevantmolecular components are identified, as well as molecules internal tothe cells involved which are involved in signal transduction pathways,it will be possible to design other therapeutics which may also be usedto inhibit induced IgE expression.

Utility

Because of their ability to effectively inhibit IgE production, thesubject anti-human CD23 antibodies which comprise human gamma-1 constantdomains, and those that may also be constructed containing gamma-3, areeffective in treating any disease wherein inhibition of IgE productionis therapeutically desirable. Such diseases include by way of exampleallergic diseases, autoimmune diseases and inflammatory disease.

Specific conditions which are potentially treatable by administration ofthe subject anti-CD23 human gamma-1/gamma-3 constant domain containingantibodies include the following:

Allergic bronchopulmonary aspergillosis; Allergic rhinitis andconjunctivitis autoimmune hemolytic anemia; Acanthosis nigricans;Allergic contact dermatitis; Addison's disease; Atopic dermatitis;Alopecia greata; Alopecia universalis; Amyloidosis; Anaphylactoidpurpura; Anaphylactoid reaction; Aplastic anemia; Angioedema,hereditary; Angioedema, idiopathic; Ankylosing spondylitis; Arteritis,cranial; Arteritis, giant cell; Arteritis, Takayasu's; Arteritis,temporal; Asthma; Ataxia-telangiectasia; Autoimmune oophoritis;Autoimmune orchitis; Autoimmune polyendocrine failure; Behcet's disease;Berger's disease; Buerger's disease; bronchitis; Bullous pemphigus;Candidiasis, chronic mucocutaneous; Caplan's syndrome; Post-myocardialinfarction syndrome; Post-pericardiotomy syndrome; Carditis; Celiacsprue; Chagas's disease; Chediak-Higashi syndrome; Churg-Straussdisease; Cogan's syndrome; Cold agglutinin disease; CREST syndrome;Crohn's disease; Cryoglobulinemia; Cryptogenic fibrosing alveolitis;Dermatitis herpetifomis; Dermatomyositis; Diabetes mellitus;Diamond-Blackfan syndrome; DiGeorge syndrome; Discoid lupuserythematosus; Eosinophilic fasciitis; Episcleritis; Drythema elevatumdiutinum; Erythema marginatum; Erythema multiforme; Erythema nodosum;Familial Mediterranean fever; Felty's syndrome; Fibrosis pulmonary;Glomerulonephritis, anaphylactoid; Glomerulonephritis, autoimmune;Glomerulonephritis, post-streptococcal; Glomerulonephritis,post-transplantation; Glomerulopathy, membranous; Goodpasture'ssyndrome; Graft-vs.-host disease; Granulocytopenia, immune-mediated;Granuloma annulare; Granulomatosis, allergic; Granulomatous myositis;Grave's disease; Hashimoto's thyroiditis; Hemolytic disease of thenewborn; Hemochromatosis, idiopathic; Henoch-Schoenlein purpura;Hepatitis, chronic active and chronic progressive; Histiocytosis_X;Hypereosinophilic syndrome; Idiopathic thrombocytopenic purpura; Job'ssyndrome; Juvenile dermatomyositis; Juvenile rheumatoid arthritis(Juvenile chronic arthritis); Kawasaki's disease; Keratitis;Keratoconjunctivitis sicca; Landry-Guillain-Barre-Strohl syndrome;Leprosy, lepromatous; Loeffler's syndrome; lupus; Lyell's syndrome; Lymedisease; Lymphomatoid granulomatosis; Mastocytosis, systemic; Mixedconnective tissue disease; Mononeuritis multiplex; Muckle-Wellssyndrome; Mucocutaneous lymph node syndrome; Mucocutaneous lymph nodesyndrome; Multicentric reticulohistiocytosis; Multiple sclerosis;Myasthenia gravis; Mycosis fungoides; Necrotizing vasculitis, systemic;Nephrotic syndrome; Overlap syndrome; Panniculitis; Paroxysmal coldhemoglobinuria; Paroxysmal nocturnal hemoglobinuria; Pemphigoid;Pemphigus; Pemphigus erythematosus; Pemphigus foliaceus; Pemphigusvulgaris; Pigeon breeder's disease; Pneumonitis, hypersensitivity;Polyarteritis nodosa; Polymyalgia rheumatic; Polymyositis; Polyneuritis,idiopathic; Portuguese familial polyneuropathies;Pre-eclampsia/eclampsia; Primary biliary cirrhosis; Progressive systemicsclerosis (Scleroderma); Psoriasis; Psoriatic arthritis; Pulmonaryalveolar proteinosis; Pulmonary fibrosis, Raynaud's phenomenon/syndrome;Reidel's thyroiditis; Reiter's syndrome, Relapsing polychrondritis;Rheumatic fever; Rheumatoid arthritis; Sarcoidosis; Scleritis;Sclerosing cholangitis; Serum sickness; Sezary syndrome; Sjogren'ssyndrome; Stevens-Johnson syndrome; Still's disease; Subacute sclerosingpanencephalitis; Sympathetic ophthalmia; Systemic lupus erythematosus;Transplant rejection; Ulcerative colitis; Undifferentiated connectivetissue disease; Urticaria, chronic; Urticaria, cold; Uveitis; Vitiligo;Weber-Christian disease; Wegener's granulomatosis; Wiskott-Aldrichsyndrome.

Of these, the preferred indications treatable or presentable byadministration of anti-CD23 antibodies include allergic rhinitis andconjunctivitis, atopic dermatitis; eczema; Job's syndrome, asthma;allergic conditions; and chronic inflammatory diseases and conditions,including CLL chronic lymphocytic leukemia, typically characterized byhigh levels of membrane CD23 and high circulating levels of sCD23(Sarfart et al., Blood 71: 94-98 (1988)).

The amount of antibody useful to produce a therapeutic effect can bedetermined by standard techniques well known to those of ordinary skillin the art. The antibodies will generally be provided by standardtechnique within a pharmaceutically acceptable buffer, and may beadministered by any desired route. Because of the efficacy of thepresently claimed anti-bodies and their tolerance by humans it ispossible to administer these antibodies repetitively in order to combatvarious diseases or disease states within a human.

One skilled in the art would be able, by routine experimentation, todetermine what an effective, non-toxic amount of antibody would be forthe purpose of effecting allergic diseases and inflammatory conditions.Generally, however, an effective dosage will be in the range of about0.05 to 100 milligrams per kilogram body weight per day.

The antibodies of the invention may be administered to a human or otheranimal in accordance with the aforementioned methods of treatment in anamount sufficient to produce such effect to a therapeutic orprophylactic degree. Such antibodies of the invention can beadministered to such human or other animal in a conventional dosage formprepared by combining the antibody of the invention with a conventionalpharmaceutically acceptable carrier or diluent according to knowntechniques. It will be recognized by one of skill in the art that theform and character of the pharmaceutically acceptable carrier or diluentis dictated by the amount of active ingredient with which it is to becombined, the route of administration and other well-known variables.

The route of administration of the antibody of the invention may beoral, parenteral, by inhalation or topical. The term parenteral as usedherein includes intravenous, intramuscular, subcutaneous, rectal,vaginal or intraperitoneal administration. The intravenous form ofparenteral administration is generally preferred.

The daily parenteral and oral dosage regimens for employing compounds ofthe invention to prophylactically or therapeutically induceimmunosuppression will generally be in the range of about 0.05 to 100,but preferably about 0.5 to 10, milligrams per kilogram body weight perday.

The antibody of the invention may also be administered by inhalation. By“inhalation” is meant intranasal and oral inhalation administration.Appropriate dosage forms for such administration, such as an aerosolformulation or a metered dose inhaler, may be prepared by conventionaltechniques. The preferred dosage amount of a compound of the inventionto be employed is generally within the range of about 10 to 100milligrams.

The antibody of the invention may also be administered topically. Bytopical administration is meant non-systemic administration and includesthe application of an antibody (or fragment thereof) compound of theinvention externally to the epidermis, to the buccal cavity andinstillation of such an antibody into the ear, eye and nose, and whereit does not significantly enter the blood stream. By systemicadministration is meant oral, intravenous, intraperitoneal andintramuscular administration. The amount of an antibody required fortherapeutic or prophylactic effect will, of course, vary with theantibody chosen, the nature and severity of the condition being treatedand the animal undergoing treatment, and is ultimately at the discretionof the physician. A suitable topical dose of an antibody of theinvention will generally be within the range of about 1 to 100milligrams per kilogram body weight daily.

Formulations

While it is possible for an antibody or fragment thereof to beadministered alone, it is preferable to present it as a pharmaceuticalformulation. The active ingredient may comprise, for topicaladministration, from 0.001% to 10% w/w, e.g., from 1% to 2% by weight ofthe formulation, although it may comprise as much as 10% w/w butpreferably not in excess of 5% w/w and more preferably from 0.1% to 1%w/w of the formulation.

The topical formulations of the present invention, comprise an activeingredient together with one or more acceptable carrier(s) therefor andoptionally any other therapeutic ingredients(s). The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of where treatment is required, such as liniments, lotions,creams, ointments or pastes, and drops suitable for administration tothe eye, ear or nose.

Drops according to the present invention may comprise sterile aqueous oroily solutions or suspensions and may be prepared by dissolving theactive ingredient in a suitable aqueous solution of a bactericidaland/or fungicidal agent and/or any other suitable preservative, andpreferably including a surface active agent. The resulting solution maythen be clarified by filtration, transferred to a suitable containerwhich is then sealed and sterilized by autoclaving or maintaining at90°-100° C. for half an hour. Alternatively, the solution may besterilized by filtration and transferred to the container by an aseptictechnique. Examples of bactericidal and fungicidal agents suitable forinclusion in the drops are phenylmercuric nitrate or acetate (0.002%),benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%).Suitable solvents for the preparation of an oily solution includeglycerol, diluted alcohol and propylene glycol.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with the aid of suitable machinery,with a greasy or non-greasy basis. The basis may comprise hydrocarbonssuch as hard, soft or liquid paraffin, glycerol, beeswax, a metallicsoap; a mucilage; an oil of natural origin such as almond, corn,arachis, castor or olive oil; wool fat or its derivatives, or a fattyacid such as stearic or oleic acid together with an alcohol such aspropylene glycol or macrogels. The formulation may incorporate anysuitable surface active agent such as an anionic, cationic or non-ionicsurface active such as sorbitan esters or polyoxyethylene derivativesthereof. Suspending agents such as natural gums, cellulose derivativesor inorganic materials such as silicaceous silicas, and otheringredients such as lanolin, may also be included.

It will be recognized by one of skill in the art that the optimalquantity and spacing of individual dosages of an antibody or fragmentthereof of the invention will be determined by the nature and extent ofthe condition being treated, the form, route and site of administration,and the particular animal being treated, and that such optimums can bedetermined by conventional techniques. It will also be appreciated byone of skill in the art that the optimal course of treatment, i.e., thenumber of doses of an antibody or fragment thereof of the inventiongiven per day for a defined number of days, can be ascertained by thoseskilled in the art using conventional course of treatment determinationtests.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following are, therefore, to be construed asmerely illustrative examples and not a limitation of the scope of thepresent invention in any way.

Capsule Composition

A pharmaceutical composition of this invention in the form of a capsuleis prepared by filling a standard two-piece hard gelatin capsule with 50mg. of an antibody or fragment thereof of the invention, in powderedform, 100 mg. of lactose, 32 mg. of talc and 8 mg. of magnesiumstearate.

Injectable Parenteral Composition

A pharmaceutical composition of this invention in a form suitable foradministration by injection is prepared by stirring 1.5 k by weight ofan antibody or fragment thereof of the invention in 10 k by volumepropylene glycol and water. The solution is sterilized by filtration.

Ointment Composition

Antibody or fragment thereof of the invention 10 g.

White soft paraffin to 100.0 g.

The antibody or fragment thereof of the invention is dispersed in asmall volume of the vehicle to produce a smooth, homogeneous product.Collapsible metal tubes are then filled with the dispersion.

Topical Cream Composition

Antibody or fragment thereof of the invention 1.0 g.

Polawax GP 200 20.0 g.

Lanolin Anhydrous 2.0 g.

White Beeswax 2.5 g.

Methyl hydroxybenzoate 0.1 g.

Distilled Water to 100.0 g.

The polawax, beeswax and lanolin are heated together at 60° C. Asolution of methyl hydroxybenzoate is added and homogenization isachieved using high speed stirring. The temperature is then allowed tofall to SOOC. The antibody or fragment thereof of the invention is thenadded and dispersed throughout, and the composition is allowed to coolwith slow speed stirring.

Topical Lotion Composition

Antibody or fragment thereof of the invention 1.0 g.

Sorbitan Monolaurate 0.6 g. Polysorbate 20 0.6 g.

Cetostearyl Alcohol 1.2 g. Glycerin 6.0 g.

Methyl Hydroxybenzoate 0.2 g.

Purified Water B.P. to 100.00 ml. (B.P.=British Pharmacopeia)

The methyl hydroxybenzoate and glycerin are dissolved in 70 ml. of thewater at 75° C. The sorbitan monolaurate, polysorbate 20 and cetostearylalcohol are melted together at 75° C. and added to the aqueous solution.The resulting emulsion is homogenized, allowed to cool with continuousstirring and the antibody or fragment thereof of the invention is addedas a suspension in the remaining water. The whole suspension is stirreduntil homogenized.

Eye Drop Composition

Antibody or fragment thereof of the invention 0.5 g.

Methyl Hydroxybenzoate 0.01 g.

Propyl Hydroxybenzoate 0.04 g.

Purified Water B.P. to 100.00 ml.

The methyl and propyl hydroxybenzoates are dissolved in 70 ml. purifiedwater at 75° C. and the resulting solution is allowed to cool. Theantibody or fragment thereof of the invention is then added, and thesolution is sterilized by filtration through a membrane filter (0.022 μmpore size), and packed aseptically into suitable sterile containers.

Composition for Administration by Inhalation

For an aerosol container with a capacity of 15-20 ml: mix 10 mg. of anantibody or fragment thereof of the invention with 0.2-0.5 k of alubricating agent, such as polysorbate 85 or oleic acid, and dispersesuch mixture in a propellant, such as freon, preferably in a combinationof (1,2 dichlorotetrafluoroethane) and difluorochloromethane and putinto an appropriate aerosol container adapted for either intranasal ororal inhalation administration. Composition for Administration byInhalation For an aerosol container with a capacity of 15-20 ml:dissolve 10 mg. of an antibody or fragment thereof of the invention inethanol (6-8 ml.), add 0.1-0.2 k of a lubricating agent, such aspolysorbate 85 or oleic acid; and disperse such in a propellant, such asfreon, preferably in combination of (1-2 dichlorotetrafluoroethane) anddifluorochloromethane, and put into an appropriate aerosol containeradapted for either intranasal or oral inhalation administration.

Parenteral Administrable Antibody Compositions

The antibodies and pharmaceutical compositions of the invention areparticularly useful for parenteral administration, i.e., subcutaneously,intramuscularly or intravenously. The compositions for parenteraladministration will commonly comprise a solution of an antibody of theinvention or a cocktail thereof dissolved in an acceptable carrier,preferably an aqueous carrier. A variety of aqueous carriers may beemployed, e.g., water, buffered water, 0.4 k saline (normal saline),0.3% glycine, and the like. The use of normal saline is preferred. Thesesolutions are sterile and generally free of particulate matter. Thesesolutions may be sterilized by conventional, well-known sterilizationtechniques. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, etc. The concentration of theantibody or fragment thereof of the invention in such pharmaceuticalformulation can vary widely. Such concentrations will be selectedprimarily based on fluid volumes, viscosities, etc., according to theparticular mode of administration selected. Generally suitableintravenous concentrations range from about one to one hundredmilligrams per milliliter.

Thus, a pharmaceutical composition of the invention for intravenousinjection could comprise 10 mL normal saline containing 40-50 mg of ananti-human CD23 antibody of the invention. Methods for preparingparenterally administrable compositions are well-known or will beapparent to those skilled in the art, and are described in more detailin, for example, Remington's Pharmaceutical Science, 15th ed., MackPublishing Company, Easton, Pa., hereby incorporated by referenceherein.

The antibodies of the invention can be lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immune globulins andart-known lyophilization and reconstitution techniques can be employed.

Depending on the intended result, the pharmaceutical composition of theinvention can be administered for prophylactic and/or therapeutictreatments. In therapeutic application, compositions are administered toa patient already suffering from a disease, in an amount sufficient tocure or at least partially arrest the disease and its complications. Inprophylactic applications, compositions containing the presentantibodies or a cocktail thereof are administered to a patient notalready in a disease state to enhance the patient's resistance.

Single or multiple administrations of the pharmaceutical compositionscan be carried out with dose levels and pattern being selected by thetreating physician. In any event, the pharmaceutical composition of theinvention should provide a quantity of the subject anti-CD23 antibodiessufficient to effectively treat the patient.

It should also be noted that the antibodies of this invention may beused for the design and synthesis of either peptide or non-peptidecompounds (mimetics) which would be useful in the same therapy as theantibody. See, e.g., Saragovi et al., Science, 253:792-795 (1991).

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various scope of the invention. Accordingly, the inventionis not limited by the appended claims.

1-39. (canceled)
 40. A method of treating a subject with a disease condition wherein inhibition of IgE is therapeutically or prophylactically beneficial comprising administering an anti-human CD23 antibody that (a) binds human CD23, (b) comprises a human gamma-1 constant region, and (c) inhibits IL-4 induced IgE expression by B-cells in vitro to a greater extent than the anti-human CD23 monoclonal antibody which lacks a human gamma-1 constant region.
 41. The method of claim 40, wherein the anti-human CD23 antibody comprises a primate antigen binding portion.
 42. The method of claim 40, wherein the anti-human CD23 antibody is a human gamma-1 monoclonal antibody.
 43. The method of claim 40, wherein the anti-human CD23 antibody comprises a rodent antigen binding portion.
 44. The method of claim 40, wherein the anti-human CD23 antibody is a humanized antibody.
 45. The method of claim 40, wherein the anti-human CD23 antibody comprises a CD23 binding affinity ranging from 0.01 nM to 1000 nM.
 46. The method of claim 45, wherein the anti-human CD23 antibody comprises a CD23 binding affinity ranging from 5 nM to 1000 nM.
 47. The method of claim 46, wherein the anti-human CD23 antibody comprises a CD23 binding affinity ranging from 100 nM to 1000 nM.
 48. The method of claim 40, wherein the anti-human CD23 antibody comprises light chain and heavy chain variable domains with sequences SEQ ID NO: 6 and SEQ ID NO: 8, respectively.
 49. The method of claim 40, wherein the anti-human CD23 antibody comprises light chain and heavy chain variable domains with sequences SEQ ID NO:2 and SEQ ID NO:4, respectively.
 50. The method of claim 40, wherein the anti-human CD23 antibody is capable of inhibiting the binding to CD23 of a monoclonal antibody which comprises light chain and heavy chain variable domains with sequences SEQ ID NO: 6 and SEQ ID NO: 8, respectively.
 51. The method of claim 40, wherein the anti-human CD23 antibody is capable of inhibiting the binding to CD23 of a monoclonal antibody which comprises light chain and heavy chain variable domains with sequences SEQ ID NO: 2 and SEQ ID NO: 4, respectively. 